Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

ABSTRACT

The present invention relates generally to the field of molecular biology and concerns a method for enhancing various yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a TCP1 or a TCP2 transcription factor, an Epsin-like polypeptide, a tRNA delta(2)-isopentenylpyrophosphate transferase (IPPT) polypeptide, or a SHORT-ROOT (SHR) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a TCP1 or TCP2 polypeptide, an Epsin-like polypeptide, a tRNA delta(2)-isopentenylpyrophosphate transferase (IPPT) polypeptide, or a SHORT-ROOT (SHR) polypeptide, which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in performing the methods of the invention.

RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.12/808,208 filed Jun. 15, 2010, which is a national stage application(under 35 U.S.C. §371) of PCT/EP2008/068129, filed Dec. 22, 2008, whichclaims benefit of European application 07123820.8, filed Dec. 20, 2007;European Application 07124011.3, filed Dec. 21, 2007; EuropeanApplication 07124036.0, filed Dec. 21, 2007; European Application07025090.7, filed Dec. 24, 2007; U.S. Provisional Application61/027,155, filed Feb. 8, 2008; U.S. Provisional Application 61/027,105,filed Feb. 8, 2008; U.S. Provisional Application 61/027,513, filed Feb.11, 2008; and U.S. Provisional Application 61/027,499, filed Feb. 11,2008. The entire content of each aforementioned application is herebyincorporated by reference in its entirety.

This application is

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)13311_(—)00090. The size ofthe text file is 645 KB, and the text file was created on Sep. 24, 2013.

The present invention relates generally to the field of molecularbiology and concerns a method for enhancing various yield-related traitsin plants by modulating expression in a plant of a nucleic acid encodinga TCP1 or a TCP2 transcription factor. The present invention alsoconcerns plants having modulated expression of a nucleic acid encoding aTCP1 or TCP2 polypeptide, which plants have enhanced yield-relatedtraits relative to corresponding wild type plants or other controlplants. The invention also provides constructs useful in the methods ofthe invention.

In another embodiment, the present invention concerns a method forimproving various plant growth characteristics by modulating expressionin a plant of a nucleic acid encoding an Epsin-like polypeptide. Thepresent invention also concerns plants having modulated expression of anucleic acid encoding an Epsin-like polypeptide, which plants haveimproved growth characteristics relative to corresponding wild typeplants or other control plants. The invention also provides constructsuseful in the methods of the invention.

In yet another embodiment, the present invention concerns a method forincreasing various plant yield-related traits by increasing expressionin the seeds of a plant, of a nucleic acid sequence encoding a tRNAdelta(2)-isopentenylpyrophosphate transferase (IPPT) polypeptide. Thepresent invention also concerns plants having increased expression inthe seeds, of a nucleic acid sequence encoding an IPPT polypeptide,which plants have increased yield-related traits relative to controlplants. The invention additionally relates to nucleic acid constructs,vectors and plants containing said nucleic acid sequences.

In further embodiment, the present invention concerns a method forenhancing yield-related traits in plants grown under conditions ofsub-optimal nutrient availability, comprising modulating expression in aplant of a nucleic acid encoding a SHORT-ROOT (SHR) polypeptide. Thepresent invention also provides a method for increasing Thousand KernelWeight (TKW) in plants relative to control plants, comprising modulatingexpression of a nucleic acid encoding an SHR polypeptide in a plantgrown under grown under non-nutrient limiting conditions. The presentinvention also concerns plants having modulated expression of a nucleicacid encoding an SHR polypeptide, which plants have enhancedyield-related traits relative to corresponding wild type plants or othercontrol plants. The invention also provides constructs useful in themethods of the invention.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuels research towards increasing theefficiency of agriculture. Conventional means for crop and horticulturalimprovements utilise selective breeding techniques to identify plantshaving desirable characteristics. However, such selective breedingtechniques have several drawbacks, namely that these techniques aretypically labour intensive and result in plants that often containheterogeneous genetic components that may not always result in thedesirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant. Such technology has the capacity to deliver crops or plantshaving various improved economic, agronomic or horticultural traits.

A trait of particular economic interest is increased yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Yield isdirectly dependent on several factors, for example, the number and sizeof the organs, plant architecture (for example, the number of branches),seed production, leaf senescence and more. Root development, nutrientuptake, stress tolerance and early vigour may also be important factorsin determining yield. Optimizing the abovementioned factors maytherefore contribute to increasing crop yield.

Seed yield is a particularly important trait, since the seeds of manyplants are important for human and animal nutrition. Crops such as corn,rice, wheat, canola and soybean account for over half the total humancaloric intake, whether through direct consumption of the seedsthemselves or through consumption of meat products raised on processedseeds. They are also a source of sugars, oils and many kinds ofmetabolites used in industrial processes. Seeds contain an embryo (thesource of new shoots and roots) and an endosperm (the source ofnutrients for embryo growth during germination and during early growthof seedlings). The development of a seed involves many genes, andrequires the transfer of metabolites from the roots, leaves and stemsinto the growing seed. The endosperm, in particular, assimilates themetabolic precursors of carbohydrates, oils and proteins and synthesizesthem into storage macromolecules to fill out the grain.

Plant biomass is yield for forage crops like alfalfa, silage corn andhay. Many proxies for yield have been used in grain crops. Chief amongstthese are estimates of plant size. Plant size can be measured in manyways depending on species and developmental stage, but include totalplant dry weight, above-ground dry weight, above-ground fresh weight,leaf area, stem volume, plant height, rosette diameter, leaf length,root length, root mass, tiller number and leaf number. Many speciesmaintain a conservative ratio between the size of different parts of theplant at a given developmental stage. These allometric relationships areused to extrapolate from one of these measures of size to another (e.g.Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at anearly developmental stage will typically correlate with plant size laterin development. A larger plant with a greater leaf area can typicallyabsorb more light and carbon dioxide than a smaller plant and thereforewill likely gain a greater weight during the same period (Fasoula &Tollenaar 2005 Maydica 50:39). This is in addition to the potentialcontinuation of the micro-environmental or genetic advantage that theplant had to achieve the larger size initially. There is a stronggenetic component to plant size and growth rate (e.g. ter Steege et al2005 Plant Physiology 139:1078), and so for a range of diverse genotypesplant size under one environmental condition is likely to correlate withsize under another (Hittalmani et al 2003 Theoretical Applied Genetics107:679). In this way a standard environment is used as a proxy for thediverse and dynamic environments encountered at different locations andtimes by crops in the field.

Another important trait for many crops is early vigour. Improving earlyvigour is an important objective of modern rice breeding programs inboth temperate and tropical rice cultivars. Long roots are important forproper soil anchorage in water-seeded rice. Where rice is sown directlyinto flooded fields, and where plants must emerge rapidly through water,longer shoots are associated with vigour. Where drill-seeding ispracticed, longer mesocotyls and coleoptiles are important for goodseedling emergence. The ability to engineer early vigour into plantswould be of great importance in agriculture. For example, poor earlyvigour has been a limitation to the introduction of maize (Zea mays L.)hybrids based on Corn Belt germplasm in the European Atlantic.

Harvest index, the ratio of seed yield to aboveground dry weight, isrelatively stable under many environmental conditions and so a robustcorrelation between plant size and grain yield can often be obtained(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes areintrinsically linked because the majority of grain biomass is dependenton current or stored photosynthetic productivity by the leaves and stemof the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa StateUniversity Press, pp 68-73). Therefore, selecting for plant size, evenat early stages of development, has been used as an indicator for futurepotential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:213). When testing for the impact of genetic differences on stresstolerance, the ability to standardize soil properties, temperature,water and nutrient availability and light intensity is an intrinsicadvantage of greenhouse or plant growth chamber environments compared tothe field. However, artificial limitations on yield due to poorpollination due to the absence of wind or insects, or insufficient spacefor mature root or canopy growth, can restrict the use of thesecontrolled environments for testing yield differences. Therefore,measurements of plant size in early development, under standardizedconditions in a growth chamber or greenhouse, are standard practices toprovide indication of potential genetic yield advantages.

A further important trait is that of improved abiotic stress tolerance.Abiotic stress is a primary cause of crop loss worldwide, reducingaverage yields for most major crop plants by more than 50% (Wang et al.,Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought,salinity, extremes of temperature, chemical toxicity, excess ordeficiency of nutrients (macroelements and/or microelements), radiationand oxidative stress. The ability to improve plant tolerance to abioticstress would be of great economic advantage to farmers worldwide andwould allow for the cultivation of crops during adverse conditions andin territories where cultivation of crops may not otherwise be possible.

Crop yield may therefore be increased by optimising one of theabove-mentioned factors.

Depending on the end use, the modification of certain yield traits maybe favoured over others. For example for applications such as forage orwood production, or bio-fuel resource, an increase in the vegetativeparts of a plant may be desirable, and for applications such as flour,starch or oil production, an increase in seed parameters may beparticularly desirable. Even amongst the seed parameters, some may befavoured over others, depending on the application. Various mechanismsmay contribute to increasing seed yield, whether that is in the form ofincreased seed size or increased seed number.

One approach to increasing yield-related traits (seed yield and/orbiomass) in plants may be through modification of the inherent growthmechanisms of a plant, such as the cell cycle or various signallingpathways involved in plant growth or in defence mechanisms.

It has now been found that various yield-related traits may be improvedin plants by modulating expression in a plant of a nucleic acid encodinga TCP1 or a TCP2 or an Epsin-like, or an SHR polypeptide as definedherein.

In another embodiment, it has been found that various yield-relatedtraits may be increased in plants relative to control plants, byincreasing expression in the seeds of a plant, of a nucleic acidsequence encoding a tRNA delta(2)-isopentenylpyrophosphate transferase(IPPT) polypeptide. The increased yield-related traits comprise one ormore of: increased early vigour, increased aboveground biomass,increased total seed yield per plant, increased total number of seeds,increased number of filled seeds, increased number of flowers perpanicles, and increased harvest index.

BACKGROUND TCP1/TCP2 Polypeptides

Transcription factors are usually defined as proteins that showsequence-specific DNA binding affinity and that are capable ofactivating and/or repressing transcription. The Arabidopsis thalianagenome codes for at least 1533 transcriptional regulators, accountingfor ˜5.9% of its estimated total number of genes (Riechmann et al.(2000) Science 290: 2105-2109). The TCP family of transcription factorsis named after its first characterized members (teosinte-branched1(TB1), cycloidea (CYC) and PCNA factor (PCF); Cubas P et al. (1999)Plant J 18(2): 215-22). In Arabidopsis thaliana, more than 20 members ofthe TCP family polypeptides have been identified, and classified basedon sequence similarity in the TCP domain into Class I (also called GroupI or PCF group) transcription factors that positively regulate geneexpression, and Class II (also called Group II or CYC-TB1 group)transcription factors that negatively regulate proliferation. All TCPtranscription factors are characterized by a non-canonical predictedbasic-Helix-Loop-Helix (bHLH), that is required for both DNA binding andhomo- and hetero-dimerization (see Cubas et al. above).

Surprisingly, it has now been found that increasing expression in aplant of a nucleic acid sequence encoding a TCP1 or a TCP2 transcriptionfactor gives plants having enhanced yield-related traits relative tocontrol plants. The particular subgroup of TCP polypeptides suitable forenhancing yield-related traits is described in detail below.

According one embodiment, there is provided a method for enhancingyield-related traits in plants relative to control plants, comprisingmodulating expression of a nucleic acid encoding a TCP1 or a TCP2polypeptide in a plant.

Epsin-Like Proteins

Eukaryotic cells possess an elaborate membrane system that functions inuptake of molecules (endocytosis) or in delivery of molecules to thecell exterior (secretory pathway). The secretory pathway leads from theendoplasmatic reticulum via the Golgi apparatus to the cell membrane.The endocytic pathway goes from the cell membrane to the cell interior.All these pathways make use of vesicles that budd off from the organellewhere they originate from and which are highly selective with respect tothe content they have and to their destination. Newly synthesisedproteins need to be transported to the different subcellular locationsor exported to the extracellular environment. Intracellular traffickingis controlled by many proteins, which are for example part of thevesicle, or assist in vesicle formation or fusion, or regulate thetrafficking or assist in selection of cargo proteins etc. Many of theseproteins are shared among plants, yeast and animals, indicating that theintracellular trafficking machinery is conserved among eukaryotes. Onesuch group of proteins is characterised by the presence of a conserved“Epsin N-Terminal Homology” (ENTH) domain. The ENTH domain is capable ofbinding to phosphatidylinositols and therefore thought to play a role intargeting these proteins to specific compartments and assist inclathrin-mediated budding. ANTH (AP180 N-Terminal homology) domains arepostulated to have a similar function as ENTH domains, but are part ofstructurally different proteins.

Epsin-like proteins all comprise an ENTH domain, and are postulated toplay similar roles in clathrin-coated vesicle formation; Epsin-likeproteins are reported to interact with various proteins (Lee et al.,Plant Physiology 143, 1561-1575, 2007; Song et al., Plant Cell 18,2258-2274, 2006).

adenylate-IPTs (AMP isopentyltransferases/ATP/ADP isopentyltransferases)

Phytohormones control plant growth and development, in response toendogenous and environmental stimuli. Examples of phytohormones includeabscisic acid, auxins, cytokinins, ethylene, gibberellins,brassinolides, salicyclic acid, jasmonates, signalling peptides, andsystemin.

In plants, naturally occurring cytokinins (CKs) constitute a group ofadenine derivatives carrying either an isopentenyl side chain(isoprenoid CKs; most abundant type) or an aromatic group (aromatic CKs;rare), and play an essential role in plant development. The first andrate-limiting step of the biosynthesis of isoprenoid CKs is catalyzed byisopentenyltransferases, which transfer the isopentenyl moiety fromdelta(2)-dimethylallyl diphosphate (DMAPP) or hydroxymethylbutenyldiphosphate (HMBDP) to position N⁶ on a conjugated adenine. Theisopentyltransferases can be subdivided into three subgroups, dependingon which conjugated adenine they utilize:

-   -   1) AMP isopentyltransferases (also named DMAPP:AMP        isopentyltransferase, EC 2.5.1.27), which preferentially use        adenosine 5′-monophosphate as acceptor molecule; typical        examples are found in phytopathogenic bacteria, such as in,        Agrobacterium tumefaciens, Pseudomonas syringae, Pseudomonas        solanacearum (Ralstonia solanacearum) and Pantoea agglomerans        (Erwinia herbicola), nitrogen-fixing symbiotic cyanobacterium        Nostoc, or slime mold Disctyostelium discoideum.    -   2) ATP/ADP isopentyltransferases (also named DMAPP:ATP/ADP        isopentyltransferase), which preferentially use adenosine        5′-triphosphate or adenosine 5′-diphosphate as acceptor        molecule; for example 8 ATP/ADP isopentyltransferases are found        in Arabidopsis thaliana (Miyawaki et al (2006) Proc Natl Acad        Sci USA 103(44): 16598-16603).    -   3) tRNA isopentyltransferases (also named DMAPP:tRNA        isopentyltransferase, or tRNA delta(2) isopentenyl pyrophosphate        transferase (IPPT), EC 2.5.1.8), which preferentially use        adenine at position 37 of certain tRNAs (located in the        cytoplasm, in the plastids and in the mitochondria), next to the        anticodon; the enzyme has been purified and the gene cloned from        bacteria, yeast, animals, and plants.

The two first subgroups (collectively named adenylate-IPTs) catalyse thedirect de novo biosynthesis of free cytokinins, essentially constitutedof isopentenyladenine (iP)-types and transzeatin (tZ)-types ofcytokinins. The third subgroup (named tRNA-IPTs or IPPTs) catalysescytokinin formation by isopentenylation of tRNA, which when degradedliberates cytokinin nucleotides, which in turn will be used tobiosynthesize cis-zeatin (cZ)-types of cytokinins. Thus, the rate oftRNA turnover also strongly determines the availability of freecytokinin nucleotides.

While tRNA is a common source of free cytokinins in prokaryotes (Koeniget al. (2002) J Bacteriol 184:1832-1842), both tRNA- and adenylate-IPTpathways contribute to cytokinin biosynthesis in seed plants (Miyawakiet al. (2006) Proc Natl Acad Sci USA 103(44): 16598-16603). However, thetRNA pathway is generally considered to be insufficient to account for asignificant source of cytokinins in seed plants. In conclusion, the twobiosynthetic pathways lead to the synthesis of different cytokinins, andin different proportions.

Both adenylate-IPTs and tRNA-IPTs have in their N-terminus the ATP/GTPP-loop binding motif (A, G)-X4-G-K-(S, T). Another well-known conservedregion specific to eucaryotic tRNA-IPTs and absent in prokaryotictRNA-IPTs, is located at the C-terminus: the Zn-finger-like motif C2H2(C-X2-C-X(12,18)-H-X5-H. The function of Zn-finger-like motif intRNA-IPTs is possibly in connection with protein-protein interactionsand nuclear localisation (Golovko et al. (2000) Gene 258: 85-93).

When an adenylate-IPT from Agrobacterium tumefaciens was constitutivelyoverexpressed in plants, or expressed at weaker or conditionally, theseshowed the typical effects of cytokinin overproduction, such asuncontrolled axillary bud growth (reduced apical dominance), theformation of small curling leaves, delayed root formation, and modifiedsenescence (for example, Luo et al. (2005) Plant Growth Regulation47:1-47, and references therein)

Transgenic Arabidopsis and canola plants expressing a bacterialadenylate-IPT under the control of a seed-specific promoter had anaverage seed yield per plant that was not significantly increasedcompared to control plants (Roeckel et al. (1997) Transgenic Res6(2):133-41).

US patent application 2006/0010515 describes transgenic Arabidopsisthaliana plants expressing an adenylate-IPT from Agrobacteriumtumefaciens using independently three cell-cycle regulated promoters,which plants have increased leaf size/vegetative mass, increased plantheight, increased branch number, increased flower and silique number.

Short Root (SHR)

Members of the GRAS gene family (an acronym based on the designations ofknown genes: GAI, RGA and SCR) encode transcriptional regulators thathave diverse functions in plant growth and development, such asgibberellin signal transduction, root radial patterning, axillarymeristem formation, phytochrome A signal transduction, andgametogenesis. Phylogenetic analysis divides the GRAS gene family intoeight subfamilies, which have distinct conserved domains and functions(Tian et al., 2004 (Plant Molecular Biology, Volume 54, Number 4, pp519-532). GRAS proteins contain a conserved region of about 350 aminoacids that can be divided in 5 motifs, found in the following order:leucine heptad repeat I, the VHIID motif, leucine heptad repeat II, thePFYRE motif and the SAW motif. SHORT ROOT, or SHR, is a member of theGRAS family of plant transcription factors and is a protein involved inroot development.

Granted patent U.S. Pat. No. 6,927,320 B1 describes SHR genes anddiscloses that SHR gene expression controls cell division of certaincell types in roots, affects the organisation of root and stem, andaffects gravitropism of aerial structures. It is suggested thatmodulation of SHR expression levels can be used to modify root andaerial structures of transgenic plants and enhance the agronomicproperties of such plants. It is also suggested that plants engineeredwith SHR overexpression may exhibit improved vigorous growthcharacteristics which may be identified by examining any of thefollowing parameters: 1. the rate of growth, 2. vegetative yield of themature plant, 3. seed or fruit yield, 4. seed or fruit weight, 5. totalnitrogen content of the plant, 6. total nitrogen content of the fruit orseed, 7. the free amino acid content of the plant, 8. the free aminoacid content of the fruit or seed, 9. the total protein content of theplant, and 10. total protein content of the fruit or seed.

SUMMARY

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a TCP1 or a TCP2 polypeptide gives plants havingenhanced yield-related traits, in particular increased yield and seedyield relative to control plants.

Also surprisingly, it has been found that modulating expression of anucleic acid encoding an Epsin-like polypeptide gives plants havingenhanced yield-related traits, in particular increased yield and/orincreased early vigour relative to control plants.

According one embodiment, there is provided a method for improving yieldrelated traits of a plant relative to control plants, comprisingmodulating expression of a nucleic acid encoding an Epsin-likepolypeptide in a plant. The improved yield related traits comprisedincreased yield and/or increased early vigour.

Furthermore, surprisingly, it has been found that increasing expressionin the seeds of a plant, of a nucleic acid sequence encoding an IPPTpolypeptide as defined herein, gives plants having increasedyield-related traits relative to control plants.

According to one embodiment, there is provided a method for increasingyield-related traits in plants relative to control plants, comprisingincreasing expression in the seeds of a plant, of a nucleic acidsequence encoding an IPPT polypeptide as defined herein. The increasedyield-related traits comprise one or more of: increased early vigour,increased aboveground biomass, increased total seed yield per plant,increased total number of seeds, increased number of filled seeds,increased number of flowers per panicles, and increased harvest index.

Furthermore, surprisingly, it has been found that modulating expressionof a nucleic acid encoding an SHR polypeptide in plants grown underconditions of sub-optimal nutrient availability gives the plantsenhanced yield-related traits relative to control plants. It has alsosurprisingly been found that modulating expression of a nucleic acidencoding an SHR polypeptide in plants grown under non-nutrient limitingconditions gives the plants increased Thousand Kernel Weight (TKW)relative to control plants.

According one embodiment of the present invention, there is thereforeprovided a method for enhancing plant yield related traits relative tocontrol plants, comprising modulating expression of a nucleic acidencoding an SHR polypeptide in a plant grown under conditions ofsub-optimal nutrient availability.

According to another embodiment of the present invention, there isprovided a method for increasing Thousand Kernel Weight (TKW) in plantsrelative to control plants, comprising modulating expression of anucleic acid encoding an SHR polypeptide in plants grown undernon-nutrient limiting conditions.

DEFINITIONS Polypeptide(s)/Protein(s)

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length, linkedtogether by peptide bonds.

Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/NucleotideSequence(s)

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are usedinterchangeably herein and refer to nucleotides, either ribonucleotidesor deoxyribonucleotides or a combination of both, in a polymericunbranched form of any length.

Control Plant(s)

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. Nullizygotes are individuals missing the transgeneby segregation. A “control plant” as used herein refers not only towhole plants, but also to plant parts, including seeds and seed parts.

Homologue(s)

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introducedinto a predetermined site in a protein. Insertions may compriseN-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than N- or C-terminalfusions, of the order of about 1 to 10 residues. Examples of N- orC-terminal fusion proteins or peptides include the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)-6-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

A substitution refers to replacement of amino acids of the protein withother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1 to 10 amino acidresidues. The amino acid substitutions are preferably conservative aminoacid substitutions. Conservative substitution tables are well known inthe art (see for example Creighton (1984) Proteins. W.H. Freeman andCompany (Eds) and Table 1 below).

TABLE 1 Examples of conserved amino acid substitutions ConservativeConservative Residue Substitutions Residue Substitutions Ala Ser LeuIle; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met;Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr GlyPro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation. Methods for the manipulation of DNA sequences to producesubstitution, insertion or deletion variants of a protein are well knownin the art. For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Derivatives

“Derivatives” include peptides, oligopeptides, polypeptides which may,compared to the amino acid sequence of the naturally-occurring form ofthe protein, such as the protein of interest, comprise substitutions ofamino acids with non-naturally occurring amino acid residues, oradditions of non-naturally occurring amino acid residues. “Derivatives”of a protein also encompass peptides, oligopeptides, polypeptides whichcomprise naturally occurring altered (glycosylated, acylated,prenylated, phosphorylated, myristoylated, sulphated etc.) ornon-naturally altered amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents or additionscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein. Furthermore, “derivatives” also includefusions of the naturally-occurring form of the protein with taggingpeptides such as FLAG, HIS6 or thioredoxin (for a review of taggingpeptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Orthologue(s)/Paralogue(s)

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

Domain

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

Motif/Consensus Sequence/Signature

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

Hybridisation

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. The hybridisation process can furthermore occur with one ofthe complementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which ahybridisation takes place. The stringency of hybridisation is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridisation buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Medium stringency conditions are when the temperatureis 20° C. below T_(m), and high stringency conditions are when thetemperature is 10° C. below T_(m). High stringency hybridisationconditions are typically used for isolating hybridising sequences thathave high sequence similarity to the target nucleic acid sequence.However, nucleic acids may deviate in sequence and still encode asubstantially identical polypeptide, due to the degeneracy of thegenetic code. Therefore medium stringency hybridisation conditions maysometimes be needed to identify such nucleic acid molecules.

The Tm is the temperature under defined ionic strength and pH, at which50% of the target sequence hybridises to a perfectly matched probe. TheT_(m) is dependent upon the solution conditions and the base compositionand length of the probe. For example, longer sequences hybridisespecifically at higher temperatures. The maximum rate of hybridisationis obtained from about 16° C. up to 32° C. below T_(m). The presence ofmonovalent cations in the hybridisation solution reduce theelectrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M (for higher concentrations, this effect maybe ignored). Formamide reduces the melting temperature of DNA-DNA andDNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, andaddition of 50% formamide allows hybridisation to be performed at 30 to45° C., though the rate of hybridisation will be lowered. Base pairmismatches reduce the hybridisation rate and the thermal stability ofthe duplexes. On average and for large probes, the Tm decreases about 1°C. per % base mismatch. The Tm may be calculated using the followingequations, depending on the types of hybrids:

1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284,1984):

-   -   T_(m)=81.5° C.+16.6×log₁₀        [Na⁺]^(a)+0.41×%[G/C^(b)]−500×[L^(c)]⁻¹−0.61×% formamide        2) DNA-RNA or RNA-RNA hybrids:    -   Tm=79.8+18.5 (log₁₀ [Na⁺]^(a))+0.58 (% G/C^(b))+11.8 (%        G/C^(b))²−820/L^(c)        3) oligo-DNA or oligo-RNA^(d) hybrids:    -   For <20 nucleotides: T_(m)=2 (l_(n))    -   For 20-35 nucleotides: T_(m)=22+1.46 (l_(n))        ^(a) or for other monovalent cation, but only accurate in the        0.01-0.04 M range.        ^(b) only accurate for % GC in the 30% to 75% range.        ^(c) L=length of duplex in base pairs.        ^(d) oligo, oligonucleotide; l_(n), =effective length of        primer=2×(no. of G/C)+(no. of NT).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase. Fornon-homologous probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridisation and which will either maintain or change the stringencyconditions.

Besides the hybridisation conditions, specificity of hybridisationtypically also depends on the function of post-hybridisation washes. Toremove background resulting from non-specific hybridisation, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridisation stringency. A positive hybridisationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridisationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridisation conditions for DNAhybrids longer than 50 nucleotides encompass hybridisation at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridisationconditions for DNA hybrids longer than 50 nucleotides encompasshybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridising nucleic acid. Whennucleic acids of known sequence are hybridised, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridisation solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

Splice Variant

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Such variants will be ones in which thebiological activity of the protein is substantially retained; this maybe achieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor predicting and isolating such splice variants are well known in theart (see for example Foissac and Schiex (2005) BMC Bioinformatics 6:25).

Allelic Variant

Alleles or allelic variants are alternative forms of a given gene,located at the same chromosomal position. Allelic variants encompassSingle Nucleotide Polymorphisms (SNPs), as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.

Gene Shuffling/Directed Evolution

Gene shuffling or directed evolution consists of iterations of DNAshuffling followed by appropriate screening and/or selection to generatevariants of nucleic acids or portions thereof encoding proteins having amodified biological activity (Castle et al., (2004) Science 304(5674):1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

Regulatory Element/Control Sequence/Promoter

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are ligated. The term“promoter” typically refers to a nucleic acid control sequence locatedupstream from the transcriptional start of a gene and which is involvedin recognising and binding of RNA polymerase and other proteins, therebydirecting transcription of an operably linked nucleic acid. Encompassedby the aforementioned terms are transcriptional regulatory sequencesderived from a classical eukaryotic genomic gene (including the TATA boxwhich is required for accurate transcription initiation, with or withouta CCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative that confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ.

A “plant promoter” comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or comprise asuitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern.

For the identification of functionally equivalent promoters, thepromoter strength and/or expression pattern of a candidate promoter maybe analysed for example by operably linking the promoter to a reportergene and assaying the expression level and pattern of the reporter genein various tissues of the plant. Suitable well-known reporter genesinclude for example beta-glucuronidase or beta-galactosidase. Thepromoter activity is assayed by measuring the enzymatic activity of thebeta-glucuronidase or beta-galactosidase. The promoter strength and/orexpression pattern may then be compared to that of a reference promoter(such as the one used in the methods of the present invention).Alternatively, promoter strength may be assayed by quantifying mRNAlevels or by comparing mRNA levels of the nucleic acid used in themethods of the present invention, with mRNA levels of housekeeping genessuch as 18S rRNA, using methods known in the art, such as Northernblotting with densitometric analysis of autoradiograms, quantitativereal-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).Generally by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts, to about 1/500,0000 transcripts per cell. Conversely, a“strong promoter” drives expression of a coding sequence at high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000transcripts per cell. Generally, by “medium strength promoter” isintended a promoter that drives expression of a coding sequence at alevel that is in all instances below that obtained under the control ofa 35S CaMV promoter.

Operably Linked

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

Constitutive Promoter

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Table 2a below gives examples of constitutivepromoters.

TABLE 2a Examples of constitutive promoters Gene Source Reference ActinMcElroy et al, Plant Cell, 2: 163-171, 1990 HMGP WO 2004/070039 CAMV 35SOdell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant J Nov;2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen et al, PlantMol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al, Plant MolBiol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol. Gen.Genet. 231: 276-285, 1992 Alfalfa H3 histone Wu et al. Plant Mol. Biol.11: 641-649, 1988 Actin 2 An et al, Plant J. 10(1); 107-121, 1996 34SFMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubisco smallU.S. Pat. No. 4,962,028 subunit OCS Leisner (1988) Proc Natl Acad SciUSA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696 SAD2Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al. (1984)Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Super promoterWO 95/14098 G-box proteins WO 94/12015

Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells ofan organism.

Developmentally-Regulated Promoter

A developmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges.

Inducible Promoter

An inducible promoter has induced or increased transcription initiationin response to a chemical (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108), environmental or physicalstimulus, or may be “stress-inducible”, i.e. activated when a plant isexposed to various stress conditions, or a “pathogen-inducible” i.e.activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”.

Examples of root-specific promoters are listed in Table 2b below:

TABLE 2b Examples of root-specific promoters Gene Source Reference RCc3Plant Mol Biol. 1995 Jan; 27(2): 237-48 Arabidopsis PHT1 Kovama et al.,2005; Mudge et al. (2002, Plant J. 31: 341) Medicago phosphate Xiao etal., 2006 transporter Arabidopsis Pyk10 Nitz et al. (2001) Plant Sci161(2): 337-346 root-expressible Tingey et al., EMBO J. 6: 1, 1987.genes tobacco auxin- Van der Zaal et al., Plant Mol. Biol. 16, induciblegene 983, 1991. β-tubulin Oppenheimer, et al., Gene 63: 87, 1988.tobacco root-specific Conkling, et al., Plant Physiol. 93: 1203, 1990.genes B. napus G1-3b gene U.S. Pat. No. 5,401,836 SbPRP1 Suzuki et al.,Plant Mol. Biol. 21: 109-119, 1993. LRX1 Baumberger et al. 2001, Genes &Dev. 15: 1128 BTG-26 Brassica US 20050044585 napus LeAMT1 (tomato)Lauter et al. (1996, PNAS 3: 8139) The LeNRT1-1 Lauter et al. (1996,PNAS 3: 8139) (tomato) class I patatin gene Liu et al., Plant Mol. Biol.153: 386-395, 1991. (potato) KDC1 Downey et al. (2000, J. Biol. Chem.275: 39420) (Daucus carota) TobRB7 gene W Song (1997) PhD Thesis, NorthCarolina State University, Raleigh, NC USA OsRAB5a (rice) Wang et al.2002, Plant Sci. 163: 273 ALF5 (Arabidopsis) Diener et al. (2001, PlantCell 13: 1625) NRT2; 1Np Quesada et al. (1997, Plant Mol. Biol. 34: 265)(N. plumbaginifolia)

A seed-specific promoter is transcriptionally active predominantly inseed tissue, but not necessarily exclusively in seed tissue (in cases ofleaky expression). The seed-specific promoter may be active during seeddevelopment and/or during germination. The seed specific promoter may bean endosperm and/or aleurone and/or embryo specific. Examples ofseed-specific promoters are shown in Table 2c to 2f below. Furtherexamples of seed-specific promoters are given in Qing Qu and Takaiwa(Plant Biotechnol. J. 2, 113-125, 2004), which disclosure isincorporated by reference herein as if fully set forth.

TABLE 2c Examples of seed-specific promoters Gene source Referenceseed-specific genes Simon et al., Plant Mol. Biol. 5: 191, 1985;Scofield et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin Pearson et al., PlantMol. Biol. 18: 235-245, 1992. legumin Ellis et al., Plant Mol. Biol. 10:203-214, 1988. glutelin (rice) Takaiwa et al., Mol. Gen. Genet. 208:15-22, 1986; Takaiwa et al., FEBS Letts. 221: 43-47, 1987. zein Matzkeet al Plant Mol Biol, 14(3): 323-32 1990 napA Stalberg et al, Planta199: 515-519, 1996. wheat LMW and HMW glutenin-1 Mol Gen Genet 216:81-90, 1989; NAR 17: 461-2, 1989 wheat SPA Albani et al, Plant Cell, 9:171-184, 1997 wheat α, β, γ-gliadins EMBO J. 3: 1409-15, 1984 barleyItr1 promoter Diaz et al. (1995) Mol Gen Genet 248(5): 592-8 barley B1,C, D, hordein Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993;Mol Gen Genet 250: 750-60, 1996 barley DOF Mena et al, The PlantJournal, 116(1): 53-62, 1998 blz2 EP99106056.7 synthetic promoterVicente-Carbajosa et al., Plant J. 13: 629-640, 1998. rice prolaminNRP33 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 ricea-globulin Glb-1 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998rice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996rice α-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33: 513-522,1997 rice ADP-glucose pyrophosphorylase Trans Res 6: 157-68, 1997 maizeESR gene family Plant J 12: 235-46, 1997 sorghum α-kafirin DeRose etal., Plant Mol. Biol 32: 1029-35, 1996 KNOX Postma-Haarsma et al, PlantMol. Biol. 39: 257-71, 1999 rice oleosin Wu et al, J. Biochem. 123: 386,1998 sunflower oleosin Cummins et al., Plant Mol. Biol. 19: 873-876,1992 PRO0117, putative rice 40S WO 2004/070039 ribosomal proteinPRO0136, rice alanine unpublished aminotransferase PRO0147, trypsininhibitor ITR1 unpublished (barley) PRO0151, rice WSI18 WO 2004/070039PRO0175, rice RAB21 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO2004/070039 α-amylase (Amy32b) Lanahan et al, Plant Cell 4: 203-211,1992; Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270, 1991cathepsin β-like gene Cejudo et al, Plant Mol Biol 20: 849-856, 1992Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 Chi26 Leah et al.,Plant J. 4: 579-89, 1994 Maize B-Peru Selinger et al., Genetics 149;1125-38, 1998

TABLE 2d examples of endosperm-specific promoters Gene source Referenceglutelin (rice) Takaiwa et al. (1986) Mol Gen Genet 208: 15-22; Takaiwaet al. (1987) FEBS Letts. 221: 43-47 zein Matzke et al., (1990) PlantMol Biol 14(3): 323-32 wheat LMW and HMW Colot et al. (1989) Mol GenGenet 216: 81-90, Anderson et al. glutenin-1 (1989) NAR 17: 461-2 wheatSPA Albani et al. (1997) Plant Cell 9: 171-184 wheat gliadins Rafalskiet al. (1984) EMBO 3: 1409-15 barley Itr1 promoter Diaz et al. (1995)Mol Gen Genet 248(5): 592-8 barley B1, C, D, hordein Cho et al. (1999)Theor Appl Genet 98: 1253-62; Muller et al. (1993) Plant J 4: 343-55;Sorenson et al. (1996) Mol Gen Genet 250: 750-60 barley DOF Mena et al,(1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem274(14): 9175-82 synthetic promoter Vicente-Carbajosa et al. (1998)Plant J 13: 629-640 rice prolamin NRP33 Wu et al, (1998) Plant CellPhysiol 39(8) 885-889 rice globulin Glb-1 Wu et al. (1998) Plant CellPhysiol 39(8) 885-889 rice globulin REB/OHP-1 Nakase et al. (1997) PlantMolec Biol 33: 513-522 rice ADP-glucose Russell et al. (1997) Trans Res6: 157-68 pyrophosphorylase maize ESR gene family Opsahl-Ferstad et al.(1997) Plant J 12: 235-46 sorghum kafirin DeRose et al. (1996) Plant MolBiol 32: 1029-35

TABLE 2e Examples of embryo specific promoters: Gene source Referencerice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 PRO0151 WO2004/070039 PRO0175 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO2004/070039

TABLE 2f Examples of aleurone-specific promoters: Gene source Referenceα-amylase Lanahan et al, Plant Cell 4: 203-211, 1992; Skriver et al,(Amy32b) Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin β- Cejudoet al, Plant Mol Biol 20: 849-856, 1992 like gene Barley Ltp2 Kalla etal., Plant J. 6: 849-60, 1994 Chi26 Leah et al., Plant J. 4: 579-89,1994 Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998

A green tissue-specific promoter as defined herein is a promoter that istranscriptionally active predominantly in green tissue, substantially tothe exclusion of any other parts of a plant, whilst still allowing forany leaky expression in these other plant parts.

Examples of green tissue-specific promoters which may be used to performthe methods of the invention are shown in Table 2g below.

TABLE 2g Examples of green tissue-specific promoters Gene ExpressionReference Maize Orthophosphate dikinase Leaf specific Fukavama et al.,2001 Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001carboxylase Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003carboxylase Rice small subunit Rubisco Leaf specific Nomura et al., 2000rice beta expansin EXBP9 Shoot specific WO 2004/070039 Pigeonpea smallsubunit Rubisco Leaf specific Panguluri et al., 2005 Pea RBCS3A Leafspecific

Another example of a tissue-specific promoter is a meristem-specificpromoter, which is transcriptionally active predominantly inmeristematic tissue, substantially to the exclusion of any other partsof a plant, whilst still allowing for any leaky expression in theseother plant parts. Examples of green meristem-specific promoters whichmay be used to perform the methods of the invention are shown in Table2h below.

TABLE 2h Examples of meristem-specific promoters Gene source Expressionpattern Reference rice OSH1 Shoot apical meristem, from Sato et al.(1996) embryo globular stage to Proc. Natl. Acad. seedling stage Sci.USA, 93: 8117-8122 Rice metallothionein Meristem specific BAD87835.1WAK1 & WAK 2 Shoot and root apical Wagner & Kohorn meristems, and inexpanding (2001) Plant Cell leaves and sepals 13(2): 303-318

Terminator

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

Modulation

The term “modulation” means in relation to expression or geneexpression, a process in which the expression level is changed by saidgene expression in comparison to the control plant, the expression levelmay be increased or decreased. The original, unmodulated expression maybe of any kind of expression of a structural RNA (rRNA, tRNA) or mRNAwith subsequent translation. The term “modulating the activity” shallmean any change of the expression of the inventive nucleic acidsequences or encoded proteins, which leads to increased yield and/orincreased growth of the plants.

Expression

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic construct. The term“expression” or “gene expression” in particular means the transcriptionof a gene or genes or genetic construct into structural RNA (rRNA, tRNA)or mRNA with or without subsequent translation of the latter into aprotein. The process includes transcription of DNA and processing of theresulting mRNA product.

Increased Expression/Overexpression

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the polypeptide ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Endogenous Gene

Reference herein to an “endogenous” gene not only refers to the gene inquestion as found in a plant in its natural form (i.e., without therebeing any human intervention), but also refers to that same gene (or asubstantially homologous nucleic acid/gene) in an isolated formsubsequently (re)introduced into a plant (a transgene). For example, atransgenic plant containing such a transgene may encounter a substantialreduction of the transgene expression and/or substantial reduction ofexpression of the endogenous gene. The isolated gene may be isolatedfrom an organism or may be manmade, for example by chemical synthesis.

Decreased Expression

Reference herein to “decreased expression” or “reduction or substantialelimination” of expression is taken to mean a decrease in endogenousgene expression and/or polypeptide levels and/or polypeptide activityrelative to control plants. The reduction or substantial elimination isin increasing order of preference at least 10%, 20%, 30%, 40% or 50%,60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reducedcompared to that of control plants. Methods for decreasing expressionare known in the art and the skilled person would readily be able toadapt the known methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

For the reduction or substantial elimination of expression an endogenousgene in a plant, a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence is required. In order to performgene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10 or fewer nucleotides, alternatively this may be as much asthe entire gene (including the 5′ and/or 3′ UTR, either in part or inwhole). The stretch of substantially contiguous nucleotides may bederived from the nucleic acid encoding the protein of interest (targetgene), or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest. Preferably, thestretch of substantially contiguous nucleotides is capable of forminghydrogen bonds with the target gene (either sense or antisense strand),more preferably, the stretch of substantially contiguous nucleotideshas, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene(either sense or antisense strand). A nucleic acid sequence encoding a(functional) polypeptide is not a requirement for the various methodsdiscussed herein for the reduction or substantial elimination ofexpression of an endogenous gene.

This reduction or substantial elimination of expression may be achievedusing routine tools and techniques. A preferred method for the reductionor substantial elimination of endogenous gene expression is byintroducing and expressing in a plant a genetic construct into which thenucleic acid (in this case a stretch of substantially contiguousnucleotides derived from the gene of interest, or from any nucleic acidcapable of encoding an orthologue, paralogue or homologue of any one ofthe protein of interest) is cloned as an inverted repeat (in part orcompletely), separated by a spacer (non-coding DNA).

In such a preferred method, expression of the endogenous gene is reducedor substantially eliminated through RNA-mediated silencing using aninverted repeat of a nucleic acid or a part thereof (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), preferably capableof forming a hairpin structure. The inverted repeat is cloned in anexpression vector comprising control sequences. A non-coding DNA nucleicacid sequence (a spacer, for example a matrix attachment region fragment(MAR), an intron, a polylinker, etc.) is located between the twoinverted nucleic acids forming the inverted repeat. After transcriptionof the inverted repeat, a chimeric RNA with a self-complementarystructure is formed (partial or complete). This double-stranded RNAstructure is referred to as the hairpin RNA (hpRNA). The hpRNA isprocessed by the plant into siRNAs that are incorporated into anRNA-induced silencing complex (RISC). The RISC further cleaves the mRNAtranscripts, thereby substantially reducing the number of mRNAtranscripts to be translated into polypeptides. For further generaldetails see for example, Grierson et al. (1998) WO 98/53083; Waterhouseet al. (1999) WO 99/53050).

Performance of the methods of the invention does not rely on introducingand expressing in a plant a genetic construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of severalwell-known “gene silencing” methods may be used to achieve the sameeffects.

One such method for the reduction of endogenous gene expression isRNA-mediated silencing of gene expression (downregulation). Silencing inthis case is triggered in a plant by a double stranded RNA sequence(dsRNA) that is substantially similar to the target endogenous gene.This dsRNA is further processed by the plant into about 20 to about 26nucleotides called short interfering RNAs (siRNAs). The siRNAs areincorporated into an RNA-induced silencing complex (RISC) that cleavesthe mRNA transcript of the endogenous target gene, thereby substantiallyreducing the number of mRNA transcripts to be translated into apolypeptide. Preferably, the double stranded RNA sequence corresponds toa target gene.

Another example of an RNA silencing method involves the introduction ofnucleic acid sequences or parts thereof (in this case a stretch ofsubstantially contiguous nucleotides derived from the gene of interest,or from any nucleic acid capable of encoding an orthologue, paralogue orhomologue of the protein of interest) in a sense orientation into aplant. “Sense orientation” refers to a DNA sequence that is homologousto an mRNA transcript thereof. Introduced into a plant would thereforebe at least one copy of the nucleic acid sequence. The additionalnucleic acid sequence will reduce expression of the endogenous gene,giving rise to a phenomenon known as co-suppression. The reduction ofgene expression will be more pronounced if several additional copies ofa nucleic acid sequence are introduced into the plant, as there is apositive correlation between high transcript levels and the triggeringof co-suppression.

Another example of an RNA silencing method involves the use of antisensenucleic acid sequences. An “antisense” nucleic acid sequence comprises anucleotide sequence that is complementary to a “sense” nucleic acidsequence encoding a protein, i.e. complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA transcriptsequence. The antisense nucleic acid sequence is preferablycomplementary to the endogenous gene to be silenced. The complementaritymay be located in the “coding region” and/or in the “non-coding region”of a gene. The term “coding region” refers to a region of the nucleotidesequence comprising codons that are translated into amino acid residues.The term “non-coding region” refers to 5′ and 3′ sequences that flankthe coding region that are transcribed but not translated into aminoacids (also referred to as 5′ and 3′ untranslated regions).

Antisense nucleic acid sequences can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid sequencemay be complementary to the entire nucleic acid sequence (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), but may also be anoligonucleotide that is antisense to only a part of the nucleic acidsequence (including the mRNA 5′ and 3′ UTR). For example, the antisenseoligonucleotide sequence may be complementary to the region surroundingthe translation start site of an mRNA transcript encoding a polypeptide.The length of a suitable antisense oligonucleotide sequence is known inthe art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10nucleotides in length or less. An antisense nucleic acid sequenceaccording to the invention may be constructed using chemical synthesisand enzymatic ligation reactions using methods known in the art. Forexample, an antisense nucleic acid sequence (e.g., an antisenseoligonucleotide sequence) may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acid sequences, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides may be used. Examples of modified nucleotidesthat may be used to generate the antisense nucleic acid sequences arewell known in the art. Known nucleotide modifications includemethylation, cyclization and ‘caps’ and substitution of one or more ofthe naturally occurring nucleotides with an analogue such as inosine.Other modifications of nucleotides are well known in the art.

The antisense nucleic acid sequence can be produced biologically usingan expression vector into which a nucleic acid sequence has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Preferably, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an operably linked antisenseoligonucleotide, and a terminator.

The nucleic acid molecules used for silencing in the methods of theinvention (whether introduced into a plant or generated in situ)hybridize with or bind to mRNA transcripts and/or genomic DNA encoding apolypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid sequence which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introducedinto a plant by transformation or direct injection at a specific tissuesite. Alternatively, antisense nucleic acid sequences can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense nucleic acid sequences can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid sequence to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid sequences canalso be delivered to cells using the vectors described herein.

According to a further aspect, the antisense nucleic acid sequence is ana-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequenceforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual b-units, the strands run parallel to each other(Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisensenucleic acid sequence may also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).

The reduction or substantial elimination of endogenous gene expressionmay also be performed using ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid sequence, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can beused to catalytically cleave mRNA transcripts encoding a polypeptide,thereby substantially reducing the number of mRNA transcripts to betranslated into a polypeptide. A ribozyme having specificity for anucleic acid sequence can be designed (see for example: Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, mRNA transcripts corresponding to a nucleic acid sequencecan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (Bartel and Szostak (1993) Science261, 1411-1418). The use of ribozymes for gene silencing in plants isknown in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by strategies asdescribed by, among others, Angell and Baulcombe ((1999) Plant J 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

Gene silencing may also occur if there is a mutation on an endogenousgene and/or a mutation on an isolated gene/nucleic acid subsequentlyintroduced into a plant. The reduction or substantial elimination may becaused by a non-functional polypeptide. For example, the polypeptide maybind to various interacting proteins; one or more mutation(s) and/ortruncation(s) may therefore provide for a polypeptide that is still ableto bind interacting proteins (such as receptor proteins) but that cannotexhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acidsequences complementary to the regulatory region of the gene (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the gene in target cells. See Helene, C.,Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad.Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.

Other methods, such as the use of antibodies directed to an endogenouspolypeptide for inhibiting its function in planta, or interference inthe signalling pathway in which a polypeptide is involved, will be wellknown to the skilled man. In particular, it can be envisaged thatmanmade molecules may be useful for inhibiting the biological functionof a target polypeptide, or for interfering with the signalling pathwayin which the target polypeptide is involved.

Alternatively, a screening program may be set up to identify in a plantpopulation natural variants of a gene, which variants encodepolypeptides with reduced activity. Such natural variants may also beused for example, to perform homologous recombination.

Artificial and/or natural microRNAs (miRNAs) may be used to knock outgene expression and/or mRNA translation. Endogenous miRNAs are singlestranded small RNAs of typically 19-24 nucleotides long. They functionprimarily to regulate gene expression and/or mRNA translation. Mostplant microRNAs (miRNAs) have perfect or near-perfect complementaritywith their target sequences. However, there are natural targets with upto five mismatches. They are processed from longer non-coding RNAs withcharacteristic fold-back structures by double-strand specific RNases ofthe Dicer family. Upon processing, they are incorporated in theRNA-induced silencing complex (RISC) by binding to its main component,an Argonaute protein. MiRNAs serve as the specificity components ofRISC, since they base-pair to target nucleic acids, mostly mRNAs, in thecytoplasm. Subsequent regulatory events include target mRNA cleavage anddestruction and/or translational inhibition. Effects of miRNAoverexpression are thus often reflected in decreased mRNA levels oftarget genes.

Artificial microRNAs (amiRNAs), which are typically 21 nucleotides inlength, can be genetically engineered specifically to negativelyregulate gene expression of single or multiple genes of interest.Determinants of plant microRNA target selection are well known in theart. Empirical parameters for target recognition have been defined andcan be used to aid in the design of specific amiRNAs, (Schwab et al.,Dev. Cell 8, 517-527, 2005). Convenient tools for design and generationof amiRNAs and their precursors are also available to the public (Schwabet al., Plant Cell 18, 1121-1133, 2006).

For optimal performance, the gene silencing techniques used for reducingexpression in a plant of an endogenous gene requires the use of nucleicacid sequences from monocotyledonous plants for transformation ofmonocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleic acidsequence from any given plant species is introduced into that samespecies. For example, a nucleic acid sequence from rice is transformedinto a rice plant. However, it is not an absolute requirement that thenucleic acid sequence to be introduced originates from the same plantspecies as the plant in which it will be introduced. It is sufficientthat there is substantial homology between the endogenous target geneand the nucleic acid to be introduced.

Described above are examples of various methods for the reduction orsubstantial elimination of expression in a plant of an endogenous gene.A person skilled in the art would readily be able to adapt theaforementioned methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

Selectable Marker (Gene)/Reporter Gene

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS or β-galactosidasewith its coloured substrates, for example X-Gal), luminescence (such asthe luciferin/luceferase system) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof). This list represents only asmall number of possible markers. The skilled worker is familiar withsuch markers. Different markers are preferred, depending on the organismand the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die). The marker genes may be removed or excised from thetransgenic cell once they are no longer needed. Techniques for markergene removal are known in the art, useful techniques are described abovein the definitions section.

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible. Naturally, these methods can also be applied tomicroorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

(a) the nucleic acid sequences encoding proteins useful in the methodsof the invention, or(b) genetic control sequence(s) which is operably linked with thenucleic acid sequence according to the invention, for example apromoter, or(c) a) and b)are not located in their natural genetic environment or have beenmodified by recombinant methods, it being possible for the modificationto take the form of, for example, a substitution, addition, deletion,inversion or insertion of one or more nucleotide residues. The naturalgenetic environment is understood as meaning the natural genomic orchromosomal locus in the original plant or the presence in a genomiclibrary. In the case of a genomic library, the natural geneticenvironment of the nucleic acid sequence is preferably retained, atleast in part. The environment flanks the nucleic acid sequence at leaston one side and has a sequence length of at least 50 bp, preferably atleast 500 bp, especially preferably at least 1000 bp, most preferably atleast 5000 bp. A naturally occurring expression cassette—for example thenaturally occurring combination of the natural promoter of the nucleicacid sequences with the corresponding nucleic acid sequence encoding apolypeptide useful in the methods of the present invention, as definedabove—becomes a transgenic expression cassette when this expressioncassette is modified by non-natural, synthetic (“artificial”) methodssuch as, for example, mutagenic treatment. Suitable methods aredescribed, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic plants are mentionedherein.

Transformation

The term “introduction” or “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. Plant tissue capableof subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA orRNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis (Arabidopsis thaliana is within thescope of the present invention not considered as a crop plant), or cropplants such as, by way of example, tobacco plants, for example byimmersing bruised leaves or chopped leaves in an agrobacterial solutionand then culturing them in suitable media. The transformation of plantsby means of Agrobacterium tumefaciens is described, for example, byHofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is knowninter alia from F. F. White, Vectors for Gene Transfer in Higher Plants;in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe inflorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent A F(1998) The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

T-DNA Activation Tagging

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),involves insertion of T-DNA, usually containing a promoter (may also bea translation enhancer or an intron), in the genomic region of the geneof interest or 10 kb up- or downstream of the coding region of a gene ina configuration such that the promoter directs expression of thetargeted gene. Typically, regulation of expression of the targeted geneby its natural promoter is disrupted and the gene falls under thecontrol of the newly introduced promoter. The promoter is typicallyembedded in a T-DNA. This T-DNA is randomly inserted into the plantgenome, for example, through Agrobacterium infection and leads tomodified expression of genes near the inserted T-DNA. The resultingtransgenic plants show dominant phenotypes due to modified expression ofgenes close to the introduced promoter.

Tilling

The term “TILLING” is an abbreviation of “Targeted Induced Local LesionsIn Genomes” and refers to a mutagenesis technology useful to generateand/or identify nucleic acids encoding proteins with modified expressionand/or activity. TILLING also allows selection of plants carrying suchmutant variants. These mutant variants may exhibit modified expression,either in strength or in location or in timing (if the mutations affectthe promoter for example). These mutant variants may exhibit higheractivity than that exhibited by the gene in its natural form. TILLINGcombines high-density mutagenesis with high-throughput screeningmethods. The steps typically followed in TILLING are: (a) EMSmutagenesis (Redei G P and Koncz C (1992) In Methods in ArabidopsisResearch, Koncz C, Chua N H, Schell J, eds. Singapore, World ScientificPublishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M,Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) InJ Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol.82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation andpooling of individuals; (c) PCR amplification of a region of interest;(d) denaturation and annealing to allow formation of heteroduplexes; (e)DHPLC, where the presence of a heteroduplex in a pool is detected as anextra peak in the chromatogram; (f) identification of the mutantindividual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (McCallum et al., (2000) NatBiotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2):145-50).

Homologous Recombination

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) butalso for crop plants, for example rice (Terada et al. (2002) Nat Biotech20(10): 1030-4; Iida and Terada (2004) Curr Opin Biotech 15(2): 132-8),and approaches exist that are generally applicable regardless of thetarget organism (Miller et al, Nature Biotechnol. 25, 778-785, 2007).

Yield

The term “yield” in general means a measurable produce of economicvalue, typically related to a specified crop, to an area, and to aperiod of time. Individual plant parts directly contribute to yieldbased on their number, size and/or weight, or the actual yield is theyield per square meter for a crop and year, which is determined bydividing total production (includes both harvested and appraisedproduction) by planted square meters. The term “yield” of a plant mayrelate to vegetative biomass (root and/or shoot biomass), toreproductive organs, and/or to propagules (such as seeds) of that plant.

Early Vigour

“Early vigour” refers to active healthy well-balanced growth especiallyduring early stages of plant growth, and may result from increased plantfitness due to, for example, the plants being better adapted to theirenvironment (i.e. optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigour alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and often betterand higher yield. Therefore, early vigour may be determined by measuringvarious factors, such as thousand kernel weight, percentage germination,percentage emergence, seedling growth, seedling height, root length,root and shoot biomass and many more.

Increase/Improve/Enhance

The terms “increase”, “improve” or “enhance” are interchangeable andshall mean in the sense of the application in case of TCP1, TCP2,Epsin-like or SHR-encoding nucleic acids or TCP1, TCP2, Epsin-like or orSHR-polypeptides at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%,preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40%more yield and/or growth in comparison to control plants as definedherein and in case of IPPT-encoding nucleic acids or IPPT-polypeptidesat least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%,more preferably 25%, 30%, 35% or 40% more yield and/or growth incomparison to control plants as defined herein.

Seed Yield

Increased seed yield may manifest itself as one or more of thefollowing: a) an increase in seed biomass (total seed weight) which maybe on an individual seed basis and/or per plant and/or per square meter;b) increased number of flowers per panicle and/or per plant; c)increased number of (filled) seeds; d) increased seed filling rate(which is expressed as the ratio between the number of filled seedsdivided by the total number of seeds); e) increased harvest index, whichis expressed as a ratio of the yield of harvestable parts, such asseeds, divided by the total biomass; f) increased thousand kernel weight(TKW) and g) increased number of primary panicles, which is extrapolatedfrom the number of filled seeds counted and their total weight. Anincreased TKW may result from an increased seed size and/or seed weight,and may also result from an increase in embryo and/or endosperm size.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter. Increased yield may also result inmodified architecture, or may occur because of modified architecture.

Greenness Index

The “greenness index” as used herein is calculated from digital imagesof plants. For each pixel belonging to the plant object on the image,the ratio of the green value versus the red value (in the RGB model forencoding color) is calculated. The greenness index is expressed as thepercentage of pixels for which the green-to-red ratio exceeds a giventhreshold. Under normal growth conditions, under salt stress growthconditions, and under reduced nutrient availability growth conditions,the greenness index of plants is measured in the last imaging beforeflowering. In contrast, under drought stress growth conditions, thegreenness index of plants is measured in the first imaging afterdrought.

Plant

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubsselected from the list comprising Acer spp., Actinidia spp., Abelmoschusspp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Erianthus sp.,Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp.,Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragariaspp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida orSoja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus),Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare),Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lensculinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffaacutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersiconpyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammeaamericana, Mangifera indica, Manihot spp., Manilkara zapota, Medicagosativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordicaspp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp.,Omithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicummiliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa,Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea,Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis,Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populusspp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyruscommunis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp.,Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp.,Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanumtuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghumbicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica,Theobroma cacao, Trifolium spp., Triticale sp., Triticosecale rimpaui,Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticumturgidum, Triticum hybernum, Triticum macha, Triticum sativum orTriticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp.,Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizaniapalustris, Ziziphus spp., amongst others.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding a TCP1 or a TCP2 polypeptide or anEpsin-like polypeptide gives plants having enhanced yield-related traitsrelative to control plants. According to a first embodiment, the presentinvention provides a method for enhancing yield-related traits in plantsrelative to control plants, comprising modulating expression in a plantof a nucleic acid encoding a TCP1 or a TCP2 polypeptide or an Epsin-likepolypeptide.

A preferred method for modulating (preferably, increasing) expression ofa nucleic acid encoding a TCP1 or a TCP2 polypeptide is by introducingand expressing in a plant a nucleic acid encoding a TCP1 or a TCP2polypeptide or an Epsin-like polypeptide.

Furthermore, surprisingly, it has been found that increasing expressionin the seeds of a plant, of a nucleic acid sequence encoding an IPPTpolypeptide as defined herein, gives plants having increasedyield-related traits relative to control plants. According to a firstembodiment, the present invention provides a method for increasingyield-related traits in plants relative to control plants, comprisingincreasing expression in the seeds of a plant, of a nucleic acidsequence encoding an IPPT polypeptide.

A preferred method for increasing expression in the seeds of a plant, ofa nucleic acid sequence encoding an IPPT polypeptide is by introducingand expressing in the seeds of a plant, a nucleic acid sequence encodingan IPPT polypeptide.

Also surprisingly, it has been found that modulating expression of anucleic acid encoding an SHR polypeptide in plants grown underconditions of sub-optimal nutrient availability gives the plantsenhanced yield-related traits relative to control plants. It has alsosurprisingly been found that modulating expression of a nucleic acidencoding an SHR polypeptide in plants grown under non nutrient-limitingconditions gives the plants increased Thousand Kernel Weight (TKW)relative to control plants.

According one embodiment, there is provided a method for enhancing yieldrelated traits relative to control plants, comprising modulatingexpression of a nucleic acid encoding an SHR polypeptide in plants grownunder conditions of sub-optimal nutrient availability.

According to another embodiment of the present invention, there isprovided a method for increasing Thousand Kernel Weight (TKW) in plantsrelative to control plants, comprising modulating expression of anucleic acid encoding an SHR polypeptide in plants grown undernon-nutrient limiting conditions.

A preferred method for modulating (preferably, increasing) expression ofa nucleic acid encoding an SHR polypeptide is by introducing andexpressing in a plant a nucleic acid encoding an SHR polypeptide.

Concerning TCP1 or a TCP2 polypeptides/genes, any reference hereinafterto a “protein useful in the methods of the invention” is taken to mean aTCP1 or a TCP2 polypeptide as defined herein. Any reference hereinafterto a “nucleic acid useful in the methods of the invention” is taken tomean a nucleic acid capable of encoding such a TCP1 or a TCP2polypeptide. The nucleic acid to be introduced into a plant (andtherefore useful in performing the methods of the invention) is anynucleic acid encoding the type of protein which will now be described,also referred to as a “TCP1 nucleic acid” or “TCP1 gene” or “TCP2nucleic acid” or “TCP2 gene”.

Regarding Epsin-like polypeptides/genes, any reference hereinafter to a“protein useful in the methods of the invention” is taken to mean anEpsin-like polypeptide as defined herein. Any reference hereinafter to a“nucleic acid useful in the methods of the invention” is taken to mean anucleic acid capable of encoding such an Epsin-like polypeptide. Thenucleic acid to be introduced into a plant (and therefore useful inperforming the methods of the invention) is any nucleic acid encodingthe type of protein which will now be described, hereafter also named“Epsin-like nucleic acid” or “Epsin-like gene”.

Concerning IPPT polypeptides/genes, any reference hereinafter to a“protein useful in the methods of the invention” is taken to mean anIPPT polypeptide as defined herein. Any reference hereinafter to a“nucleic acid sequence useful in the methods of the invention” is takento mean a nucleic acid sequence capable of encoding such an IPPTpolypeptide. The nucleic acid sequence to be introduced into a plant(and therefore useful in performing the methods of the invention) is anynucleic acid sequence encoding the type of polypeptide, which will nowbe described, hereafter also named “IPPT nucleic acid sequence” or “IPPTgene”.

Regarding SHR polypeptides/genes, any reference hereinafter to a“protein useful in the methods of the invention” is taken to mean an SHRpolypeptide as defined herein. Any reference hereinafter to a “nucleicacid useful in the methods of the invention” is taken to mean a nucleicacid capable of encoding such an SHR polypeptide. The nucleic acid to beintroduced into a plant (and therefore useful in performing the methodsof the invention) is any nucleic acid encoding the type of protein whichwill now be described, hereinafter also named “SHR nucleic acid” or “SHRgene”.

A “TCP1 polypeptide” as defined herein refers to any polypeptidecomprising:

(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of any one of the sequences indicated in FIG. 1; and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain A of any one of the sequences indicated in FIG. 1; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain B of any one of the sequences indicated in FIG. 1; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain C of any one of the sequences indicated in FIG. 1.

According to a preferred embodiment, the TCP1 polypeptide comprises:

(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of the sequence represented by Ms_TCP_sugar in FIG. 1;and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain A of the sequence represented by Ms_TCP_sugar in FIG. 1; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain B of the sequence represented by Ms_TCP_sugar in FIG. 1; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain C of the sequence represented by Ms_TCP_sugar in FIG. 1.

A “TCP2 polypeptide” as defined herein refers to any polypeptidecomprising:

(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of any of the sequences indicated in FIG. 2; and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 1 of any of the sequences indicated in FIG. 2; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 2 of any of the sequences indicated in FIG. 2; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 3 of any of the sequences indicated in FIG. 2.

According to a preferred embodiment, the TCP2 polypeptide comprises:

(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of the sequence represented by Mt_TCP2_sugar in FIG. 2;and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 1 of the sequence represented by Mt_TCP2_sugar in FIG. 2; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 2 of the sequence represented by Mt_TCP2_sugar in FIG. 2; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 3 of the sequence represented by Mt_TCP2_sugar in FIG. 2.

In addition, the TCP2 polypeptide may comprise any one or both of:

(v) a domain having in increasing order of preference at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 4 of any of the sequences indicated in FIG. 2;(vi) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 5 of any of the sequences indicated in FIG. 2.

Preferably, Domain 4 has in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 4 of the sequence represented by Mt_TCP2_sugar in FIG. 2.

Preferably, Domain 5 has in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 5 of the sequence represented by Mt_TCP2_sugar in FIG. 2.

The TCP1 or TCP2 protein has in increasing order of preference at least25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acidrepresented by SEQ ID NO: 2 or SEQ ID NO: 4 respectively. The overallsequence identity is determined using a global alignment algorithm, suchas the Needleman Wunsch algorithm in the program GAP (GCG WisconsinPackage, Accelrys), preferably with default parameters. Compared tooverall sequence identity, the sequence identity will generally behigher when only conserved domains or motifs are considered.

Preferably, the TCP1 or TCP2 polypeptide sequence which when used in theconstruction of a TCP phylogenetic tree, such as the one depicted inFIG. 2, clusters with the group of TCP1 or TCP2 polypeptides comprisingthe amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4rather than with any other group.

A “Epsin-like polypeptide” as defined herein refers to any polypeptidecomprising an ENTH domain (SMART accession SM00273) in its N-terminalhalf. The ENTH domain is known in the art and is described in theInterPro database: The ENTH (Epsin N-terminal homology) domain isapproximately 150 amino acids in length and is always found located atthe N-termini of proteins. The domain forms a compact globularstructure, composed of 9 alpha-helices connected by loops of varyinglength. The general topology is determined by three helical hairpinsthat are stacked consecutively with a right hand twist. An N-terminalhelix folds back, forming a deep basic groove that forms the bindingpocket for the Ins(1,4,5)P3 ligand. The ligand is coordinated byresidues from surrounding alpha-helices and all three phosphates aremultiply coordinated. The coordination of Ins(1,4,5)P3 suggests thatENTH is specific for particular head groups. Proteins containing thisdomain have been found to bind PtdIns(4,5)P2 and PtdIns(1,4,5)P3suggesting that the domain may be a membrane interacting module. Themain function of proteins containing this domain appears to be to act asaccessory clathrin adaptors in endocytosis, Epsin is able to recruit andpromote clathrin polymerisation on a lipid monolayer, but may haveadditional roles in signalling and actin regulation. Epsin causes astrong degree of membrane curvature and tubulation, even fragmentationof membranes with a high PtdIns(4,5)P2 content. Epsin binding tomembranes facilitates their deformation by insertion of the N-terminalhelix into the outer leaflet of the bilayer, pushing the head groupsapart. This would reduce the energy needed to curve the membrane into avesicle, making it easier for the clathrin cage to fix and stabilise thecurved membrane. This points to a pioneering role for epsin in vesiclebudding as it provides both a driving force and a link between membraneinvagination and clathrin polymerisation (annotation IPR013809).

Preferably, the Epsin-like polypeptide useful in the methods of thepresent invention furthermore comprises two or more of the followingmotifs:

Motif 1: (SEQ ID NO: 48)(V/I)(L/R)(D/E)AT(S/D/N)(N/D/E/S)E(P/S)WGPHG(T/S/E)Preferably, Motif 1 is: (V/I)LDAT(S/D/N)(N/D)E(P/S)WGPHG(T/S)More preferably, Motif 1 is VLDATDNEPWGPHGT Motif 2: (SEQ ID NO: 49)F(Q/E)(Y/F)(I/L/V/R/K)(D/E)(S/P/A)(S/G/N/Q/R)G(R/K)D(Q/V/A/H/E)G(S/N/L/I/V)NVRPreferably, Motif 2 is:F(Q/E)(Y/F)(I/L/V)(D/E)(S/P)(S/G/N)G(R/K)D(Q/V/A)G(S/N/L/I)NVRMore preferably, Motif 2 is FEYVEPNGKDVGINVR Motif 3: (SEQ ID NO: 50)(E/S/A/Q)(V/I/E/A)R(Q/E/D/N)KA(A/L/V/E)(A/V/S/R/K)(N/T)(R/A)(D/E/N/G)KPreferably, Motif 3 is:(E/S/A)(V/I)R(Q/E/D/N)KA(A/L/V)(A/V/S)(N/T)R(D/E/N)KMore preferably, Motif 3 is EIRDKAVANRNK Motif 4: (SEQ ID NO: 51)WAD(T/S)LSRGL(V/I) Preferably, Motif 4 is: WADSLSRGLI Motif 5:(SEQ ID NO: 52) L(A/S)D(I/V)G(I/V)(D/V)(F/G)(D/E/P/G)Preferably, Motif 5 is: LADVGVVGD

In addition to the previous motifs, the protein useful in the methods ofthe present invention preferably also comprises in its native form oneor more of the following motifs:

Motif 6 (a to c): one of the following tetrapeptides: GGYG, GSYG or GGYD(SEQ ID NO: 53, 54, 55)Motif 7 (a to d): one of the following tetrapeptides: SAAS, SSAS, SSAP,or SSAT (SEQ ID NO: 56, 57, 58, 59)Motif 8 (a to e): one of the following tetrapeptides: DEFD, DFFD, DDDF,EDDF, or DDFD (SEQ ID NO: 60, 61, 62, 63, 64)

Alternatively, the homologue of an Epsin-like protein has in increasingorder of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overallsequence identity to the amino acid represented by SEQ ID NO: 44,provided that the homologous protein comprises two or more of theconserved motifs as outlined above. The overall sequence identity isdetermined using a global alignment algorithm, such as the NeedlemanWunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys),preferably with default parameters. Compared to overall sequenceidentity, the sequence identity will generally be higher when onlyconserved domains or motifs are considered. For example, when the ENTHdomain is compared among the Epsin-like polypeptides, the sequenceidentity will be much higher compared to the overall sequence identity.

Preferably, the polypeptide sequence which when used in the constructionof a phylogenetic tree, such as the one depicted in FIG. 3 (Holstein andOliviusson, Protoplasma 226, 13-21, 2005), clusters with the group ofEpsin-like polypeptides comprising the amino acid sequence representedby SEQ ID NO: 44 rather than with any other group.

An “IPPT polypeptide” as defined herein refers to any polypeptidecomprising (i) a tRNA isopentenyltransferase domain with an InterProaccession IPR002627; and (ii) an N-terminal ATP/GTP-binding site motif A(P-loop).

Alternatively or additionally, an “IPPT polypeptide” as defined hereinrefers to any polypeptide sequence having (i) in increasing order ofpreference at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more aminoacid sequence identity to an N-terminal ATP/GTP-binding site motif A(P-loop) as represented by SEQ ID NO: 199; and having in increasingorder of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% ormore amino acid sequence identity to one or more of: (ii) Conservedmotif I DSR(Q/L)(V/L/I) as represented by SEQ ID NO: 200; or (ii)Conserved motif II (N/D/S/T)(I/V)GTAKP(T/S) as represented by SEQ ID NO:201; or (iii) Conserved motif III L(V/A/I)GG(S/T)GLY as represented bySEQ ID NO:202; or (iv) Conserved motif IV F/Y/L)AK(R/K/Q)Q(R/K/M)TWFR asrepresented by SEQ ID NO:203.

Alternatively or additionally, an “IPPT polypeptide” as defined hereinrefers to any polypeptide having in increasing order of preference atleast 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to the IPPT polypeptide asrepresented by SEQ ID NO: 144 or to any of the polypeptide sequencesgiven in Table A4 herein.

Alternatively or additionally, an “IPPT polypeptide” is capable ofcomplementing a yeast mod 5 mutant strain which lacks endogenous IPPTactivity, or is capable of complementing an E. coli miaA mutant strainwhich lacks endogenous IPPT activity.

An “SHR polypeptide” as defined herein refers to any full lengthpolypeptide which when used in the construction of a GRAS phylogenetictree, such as the one depicted in FIG. 14, clusters with the group ofSHR polypeptides comprising the amino acid sequence represented by SEQID NO: 209 rather than with any other group.

SHR polypeptides, being members of the GRAS family of planttranscription factors, may comprise features typical of the GRAS genefamily. Such typical features include a highly conserved C-terminalregion, but variable N-terminal region. The highly conserved C-terminalregion comprises five distinct motifs, typically found in the followingorder:

1. leucine heptad repeat (LHR1),2. VHIID motif,3. leucine heptad repeat II (LHR II),4. PFYRE motif, and5. SAW motif.

LHR I appears to consist of two repeat units that are separated by aspacer that often contains a proline residue, known to disruptalpha-helical structures. The two units within LHR I are not in phasewith each other. LHR IA is similar to LHRs found in other proteins,consisting of between three to five regular heptads. LHR IB is shorter,usually consisting of only two such repeats. In LHR II, specific leucineheptad repeats can be identified in this region in nearly all members ofthe GRAS family, the number of repeats is small, usually two or three.

The VHIID sequence is readily recognizable in all members of the family,although it is not absolutely

conserved: substitutions of valine, isoleucine and leucine at the 1, 3and 4 positions yield a number of permutations. Within the larger regionthat we term the VHIID motif, the P-N-H-D-Q-L residues are absolutelyconserved. The spacing between the proline and asparagine residues isidentical among all members, as is the spacing between the histidine,aspartate, glutamine and leucine residues. The VHIID motif is bounded atits C-terminus by a conserved sequence referred to as LRITG forsimplicity.

Most of the deviations from this consensus sequence representconservative changes.

In the PFYRE motif, P is absolutely conserved. Within the PFYRE domain,the sequences are largely co-linear and portions of this region show ahigh degree of sequence similarity among all members of the GRAS family.

The SAW motif is characterized by three pairs of absolutely conservedresidues: R-E, W-G and W-W. The W-W pair found nearly at the C-terminusof these sequences shows absolute conservation of spacing, as does theW-G pair.

In addition to an SHR polypeptide clustering with other SHR polypeptidesin a GRAS phylogenetic tree, preferably, the C-terminal region an SHRpolypeptide useful in the methods of the invention has in increasingorder of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%overall sequence identity to the C-terminal region of the amino acidrepresented by SEQ ID NO: 209.

The overall sequence identity is determined using a global alignmentalgorithm, such as the Needleman Wunsch algorithm in the program GAP(GCG Wisconsin Package, Accelrys), preferably with default parameters.Compared to overall sequence identity, the sequence identity willgenerally be higher when only conserved domains or motifs areconsidered.

The term “domain” and “motif” is defined in the “definitions” sectionherein. Specialist databases exist for the identification of domains,for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Batemanet al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of toolsfor in silico analysis of protein sequences is available on the ExPASyproteomics server (Swiss Institute of Bioinformatics (Gasteiger et al.,ExPASy: the proteomics server for in-depth protein knowledge andanalysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs mayalso be identified using routine techniques, such as by sequencealignment.

Analysis of the polypeptide sequence of SEQ ID NO: 144 is presentedbelow in Example 4 herein. For example, an IPPT polypeptide asrepresented by SEQ ID NO: 144 comprises a tRNA isopentenyltransferasedomain with an InterPro accession IPR002627. Domains may also beidentified using routine techniques, such as by sequence alignment. Analignment of the polypeptides of Table A4 herein, is shown in FIG. 3.Such alignments are useful for identifying the most conserved domains ormotifs between the IPPT polypeptides, such as the Conserved motifs asrepresented by SEQ ID NO: 200 to 203 (comprised in SEQ ID NO: 144).

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information(NCBI). Homologues may readily be identified using, for example, theClustalW multiple sequence alignment algorithm (version 1.83), with thedefault pairwise alignment parameters, and a scoring method inpercentage. Global percentages of similarity and identity may also bedetermined using one of the methods available in the MatGAT softwarepackage (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29.MatGAT: an application that generates similarity/identity matrices usingprotein or DNA sequences.). Minor manual editing may be performed tooptimise alignment between conserved motifs, as would be apparent to aperson skilled in the art. Furthermore, instead of using full-lengthsequences for the identification of homologues, specific domains mayalso be used. The sequence identity values may be determined over theentire nucleic acid or amino acid sequence or over selected domains orconserved motif(s), using the programs mentioned above using the defaultparameters. For local alignments, the Smith-Waterman algorithm isparticularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1);195-7).

Furthermore, TCP 1 and TCP2 polypeptides (at least in their native form)typically have DNA binding activity. Tools and techniques for measuringDNA binding activity are well known in the art. Further details areprovided in Example 6.

In addition, TCP 1 and TCP2 polypeptides, when expressed in riceaccording to the methods of the present invention as outlined in theExamples section, give plants having increased yield related traits, inparticular increased seed yield.

Furthermore, Epsin-like polypeptides (at least in their native form)typically have lipid binding activity. Tools and techniques formeasuring lipid binding activity are well known in the art. For example,lipid binding by the ENTH domain is described by Hom et al. (J. Mol.Biol. 373, 412-423, 2007). Further details are provided in Example 6.

In addition, Epsin-like polypeptides, when expressed in rice accordingto the methods of the present invention as outlined in Examples 7 and 8,give plants having increased yield related traits, in particular one ormore of increased total weight of seeds, fill rate, total number ofseeds and number of filled seeds.

Example 3 herein describes in Table B4 the percentage identity betweenthe IPPT polypeptide as represented by SEQ ID NO: 144 and the IPPTpolypeptides listed in Table A4, which can be as low as 39% amino acidsequence identity.

The task of protein subcellular localisation prediction is important andwell studied. Knowing a protein's localisation helps elucidate itsfunction. Experimental methods for protein localization range fromimmunolocalization to tagging of proteins using green fluorescentprotein (GFP) or beta-glucuronidase (GUS). Such methods are accuratealthough labor-intensive compared with computational methods. Recentlymuch progress has been made in computational prediction of proteinlocalisation from sequence data. Among algorithms well known to a personskilled in the art are available at the ExPASy Proteomics tools hostedby the Swiss Institute for Bioinformatics, for example, PSort, TargetP,ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM,and others.

Furthermore, IPPT polypeptides useful in the methods of the presentinvention (at least in their native form) typically are capable oftransferring the isopentenyl moiety from delta(2)-dimethylallyldiphosphate (DMAPP) or hydroxymethylbutenyl diphosphate (HMBDP) to anadenine at position 37 of certain tRNAs. Many assays exist to measuresuch enzymatic activity, including complementation assays of a yeaststrain with defective endogenous IPPT activity (encoded by the MOD 5gene; Golovko et al. (2002) Plant Molec Biol 49: 161-169),complementation assays of an E. coli strain with defective endogenousIPPT activity (encoded by the miaA gene; Dihanich et al. (1987) Mol CellBiol 7: 177-184), or quantification of cytokinins in tRNA (Gray et al.(1996) Plant Physiol 110: 431-438, Miyawaki et al. (2006) Proc Natl AcadSCi USA 103(44): 16598-16603).

In addition, SHR polypeptides, when expressed in rice grown underconditions of sub-optimal nutrient availability gives the plantsenhanced yield-related traits relative to control plants. SHRpolypeptides when expressed in rice grown under non-nutrient limitingconditions gives the plants increased Thousand Kernel Weight (TKW) inplants relative to control plants.

Concerning TCP1 and TCP2, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 1, encoding the polypeptide sequence of SEQ ID NO: 2 and bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 3, encoding the polypeptide sequence of SEQ ID NO: 4. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyTCP1-encoding or TCP2-encoding nucleic acid, or using a TCP1 or TCP2polypeptide as defined herein.

Concerning TCP1 and TCP2, examples of nucleic acids encoding TCP1 andTCP2 polypeptides are given in Example 1 herein. Such nucleic acids areuseful in performing the methods of the invention. The amino acidsequences encoded by the nucleic acid sequences given in Example 1 areexample sequences of orthologues and paralogues of the TCP 1 polypeptiderepresented by SEQ ID NO: 2, and the amino acid sequences encoded by thenucleic acid sequences given in Example 1 are example sequences oforthologues and paralogues of the TCP 2 polypeptide represented by SEQID NO: 4, the terms “orthologues” and “paralogues” being as definedherein. Further orthologues and paralogues may readily be identified byperforming a so-called reciprocal blast search. Typically, this involvesa first BLAST involving BLASTing a query sequence (for example using anyof the sequences listed in Example 1) against any sequence database,such as the publicly available NCBI database. BLASTN or TBLASTX (usingstandard default values) are generally used when starting from anucleotide sequence, and BLASTP or TBLASTN (using standard defaultvalues) when starting from a protein sequence. The BLAST results mayoptionally be filtered. The full-length sequences of either the filteredresults or non-filtered results are then BLASTed back (second BLAST)against sequences from the organism from which the query sequence isderived (where the query sequence is SEQ ID NO: 1 to SEQ ID NO: 4, thesecond BLAST would therefore be against Medicago sequences). The resultsof the first and second BLASTs are then compared. A paralogue isidentified if a high-ranking hit from the first blast is from the samespecies as from which the query sequence is derived, a BLAST back thenideally results in the query sequence amongst the highest hits; anorthologue is identified if a high-ranking hit in the first BLAST is notfrom the same species as from which the query sequence is derived, andpreferably results upon BLAST back in the query sequence being among thehighest hits.

Concerning Epsin-like-sequences, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 43, encoding the polypeptide sequence of SEQ ID NO: 44. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyEpsin-like-encoding nucleic acid or Epsin-like polypeptide as definedherein.

Concerning Epsin-like-sequences, examples of nucleic acids encodingEpsin-like polypeptides are given in Table A3 of Example 1 herein. Suchnucleic acids are useful in performing the methods of the invention. Theamino acid sequences given in Table A3 of Example 1 are examplesequences of orthologues and paralogues of the Epsin-like polypeptiderepresented by SEQ ID NO: 44, the terms “orthologues” and “paralogues”being as defined herein. Further orthologues and paralogues may readilybe identified by performing a so-called reciprocal blast search.Typically, this involves a first BLAST involving BLASTing a querysequence (for example using any of the sequences listed in Table A3 ofExample 1) against any sequence database, such as the publicly availableNCBI database. BLASTN or TBLASTX (using standard default values) aregenerally used when starting from a nucleotide sequence, and BLASTP orTBLASTN (using standard default values) when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 43 or SEQ ID NO: 44, the second BLAST would therefore beagainst Arabidopsis sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first blast is from the same species as from which thequery sequence is derived, a BLAST back then ideally results in thequery sequence amongst the highest hits; an orthologue is identified ifa high-ranking hit in the first BLAST is not from the same species asfrom which the query sequence is derived, and preferably results uponBLAST back in the query sequence being among the highest hits.

Concerning IPPT, the present invention is illustrated by transformingplants with the nucleic acid sequence represented by SEQ ID NO: 143,encoding the IPPT polypeptide sequence of SEQ ID NO: 144. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anynucleic acid sequence encoding an IPPT polypeptide as defined herein.

Concerning IPPT, examples of nucleic acid sequences encoding IPPTpolypeptides are given in Table A4 of Example 1 herein. Such nucleicacid sequences are useful in performing the methods of the invention.The polypeptide sequences given in Table A4 of Example 1 are examplesequences of orthologues and paralogues of the IPPT polypeptiderepresented by SEQ ID NO: 144, the terms “orthologues” and “paralogues”being as defined herein. Further orthologues and paralogues may readilybe identified by performing a so-called reciprocal blast search.Typically, this involves a first BLAST involving BLASTing a querysequence (for example using any of the sequences listed in Table A4 ofExample 1) against any sequence database, such as the publicly availableNCBI database. BLASTN or TBLASTX (using standard default values) aregenerally used when starting from a nucleotide sequence, and BLASTP orTBLASTN (using standard default values) when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 143 or SEQ ID NO: 144, the second BLAST would therefore beagainst Synechococcus sp. PCC 7942 sequences). The results of the firstand second BLASTs are then compared. A paralogue is identified if ahigh-ranking hit from the first blast is from the same species as fromwhich the query sequence is derived, a BLAST back then ideally resultsin the query sequence amongst the highest hits; an orthologue isidentified if a high-ranking hit in the first BLAST is not from the samespecies as from which the query sequence is derived, and preferablyresults upon BLAST back in the query sequence being among the highesthits.

Concerning SHR, the present invention is illustrated by transformingplants with the nucleic acid sequence represented by SEQ ID NO: 208,encoding the polypeptide sequence of SEQ ID NO: 209. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anySHR-encoding nucleic acid or SHR polypeptide as defined herein.

Concerning SHR, examples of nucleic acids encoding SHR polypeptides aregiven in Table A5 of Example 1 herein. Such nucleic acids are useful inperforming the methods of the invention. The amino acid sequences givenin Table A5 of Example 1 are example sequences of orthologues andparalogues of the SHR polypeptide represented by SEQ ID NO: 209, theterms “orthologues” and “paralogues” being as defined herein. Furtherorthologues and paralogues may readily be identified by performing aso-called reciprocal blast search. Typically, this involves a firstBLAST involving BLASTing a query sequence (for example using any of thesequences listed in Table A5 of Example 1) against any sequencedatabase, such as the publicly available NCBI database. BLASTN orTBLASTX (using standard default values) are generally used when startingfrom a nucleotide sequence, and BLASTP or TBLASTN (using standarddefault values) when starting from a protein sequence. The BLAST resultsmay optionally be filtered. The full-length sequences of either thefiltered results or non-filtered results are then BLASTed back (secondBLAST) against sequences from the organism from which the query sequenceis derived (where the query sequence is SEQ ID NO: 208 or SEQ ID NO:209, the second BLAST would therefore be against Arabidopsis sequences).The results of the first and second BLASTs are then compared. Aparalogue is identified if a high-ranking hit from the first blast isfrom the same species as from which the query sequence is derived, aBLAST back then ideally results in the query sequence amongst thehighest hits; an orthologue is identified if a high-ranking hit in thefirst BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Nucleic acid variants may also be useful in practising the methods ofthe invention. Examples of such variants include nucleic acids encodinghomologues and derivatives of any one of the amino acid sequencesencoded by the nucleic acid sequences given in table A of Example 1, theterms “homologue” and “derivative” being as defined herein. Also usefulin the methods of the invention are nucleic acids encoding homologuesand derivatives of orthologues or paralogues of any one of the aminoacid sequences encoded by the nucleic acid sequences given in table A ofExample 1. Homologues and derivatives useful in the methods of thepresent invention have substantially the same biological and functionalactivity as the unmodified protein from which they are derived.

Further nucleic acid variants useful in practising the methods of theinvention include portions of nucleic acids encoding TCP1 or TCP2,Epsin-like, IPPT or SHR polypeptides, nucleic acids hybridising tonucleic acids encoding TCP1 or TCP2, Epsin-like, IPPT or SHRpolypeptides, splice variants of nucleic acids encoding TCP1 or TCP2,Epsin-like, IPPT or SHR polypeptides, allelic variants of nucleic acidsencoding TCP1 or TCP2, Epsin-like, IPPT or SHR polypeptides and variantsof nucleic acids encoding TCP1 or TCP2, Epsin-like, IPPT or SHRpolypeptides obtained by gene shuffling. The terms hybridising sequence,splice variant, allelic variant and gene shuffling are as describedherein.

Nucleic acids encoding TCP1 or TCP2, Epsin-like, or IPPT need not befull-length nucleic acids, since performance of the methods of theinvention does not rely on the use of full-length nucleic acidsequences. According to the present invention, there is provided amethod for enhancing yield-related traits in plants, comprisingintroducing and expressing in a plant a portion of any one of thenucleic acid sequences encoded by the nucleic acid sequences given intable A of Example 1, or a portion of a nucleic acid encoding anorthologue, paralogue or homologue of any of the amino acid sequencesencoded by the nucleic acid sequences given in Example 1.

Nucleic acids encoding SHR polypeptides need not be full-length nucleicacids, since performance of the methods of the invention does not relyon the use of full-length nucleic acid sequences. According to thepresent invention, there is provided a method for enhancingyield-related traits in plants grown under conditions of sub-optimalnutrient availability, comprising introducing and expressing in a planta portion of any one of the nucleic acid sequences given in Table A5 ofExample 1, or a portion of a nucleic acid encoding an orthologue,paralogue or homologue of any of the amino acid sequences given in TableA5 of Example 1. There is also provided a method for increasing TKW inplants grown under non-nutrient limiting conditions, comprisingintroducing and expressing in a plant a portion of any one of thenucleic acid sequences given in Table A5 of Example 1, or a portion of anucleic acid encoding an orthologue, paralogue or homologue of any ofthe amino acid sequences given in Table A5 of Example 1.

A portion of a nucleic acid may be prepared, for example, by making oneor more deletions to the nucleic acid. The portions may be used inisolated form or they may be fused to other coding (or non-coding)sequences in order to, for example, produce a protein that combinesseveral activities. When fused to other coding sequences, the resultantpolypeptide produced upon translation may be bigger than that predictedfor the protein portion.

Concerning TCP1 or TCP2, portions useful in the methods of theinvention, encode a TCP1 or TCP2 polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesencoded by the nucleic acid sequences given in Example 1. Preferably,the portion is a portion of any one of the nucleic acids given inExample 1, or is a portion of a nucleic acid encoding an orthologue orparalogue of any one of the amino acid sequences encoded by the nucleicacid sequences given in Example 1. Preferably the portion is at least500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500 consecutive nucleotides inlength, the consecutive nucleotides being of any one of the nucleic acidsequences given in Example 1, or of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences encodedby the nucleic acid sequences given in Example 1. Most preferably theportion is a portion of the nucleic acid of SEQ ID NO: 1 or SEQ ID NO:3. Preferably, the portion encodes a fragment of an amino acid sequencewhich, when used in the construction of a phylogenetic tree, such as theone depicted in FIG. 1 or 2, clusters with the group of TCP1 or TCP2polypeptides comprising the amino acid sequence represented by SEQ IDNO: 2 or SEQ ID NO: 4 rather than with any other group.

Concerning Epsin-like sequences, portions useful in the methods of theinvention, encode an Epsin-like polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A3 of Example 1. Preferably, the portion is a portion ofany one of the nucleic acids given in Table A3 of Example 1, or is aportion of a nucleic acid encoding an orthologue or paralogue of any oneof the amino acid sequences given in Table A3 of Example 1. Preferablythe portion is at least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150,2200, 2250, 2300 consecutive nucleotides in length, the consecutivenucleotides being of any one of the nucleic acid sequences given inTable A3 of Example 1, or of a nucleic acid encoding an orthologue orparalogue of any one of the amino acid sequences given in Table A3 ofExample 1. Most preferably the portion is a portion of the nucleic acidof SEQ ID NO: 43. Preferably, the portion encodes a fragment of an aminoacid sequence which, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 3 (Holstein and Oliviusson,Protoplasma 226, 13-21, 2005), clusters with the group of Epsin-likepolypeptides comprising the amino acid sequence represented by SEQ IDNO: 44 rather than with any other group.

Concerning IPPT, portions useful in the methods of the invention, encodean IPPT polypeptide as defined herein, and have substantially the samebiological activity as the polypeptide sequences given in Table A4 ofExample 1. Preferably, the portion is a portion of any one of thenucleic acid sequences given in Table A4 of Example 1, or is a portionof a nucleic acid sequence encoding an orthologue or paralogue of anyone of the polypeptide sequences given in Table A4 of Example 1.Preferably the portion is, in increasing order of preference at least400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 920 or moreconsecutive nucleotides in length, the consecutive nucleotides being ofany one of the nucleic acid sequences given in Table A4 of Example 1, orof a nucleic acid sequence encoding an orthologue or paralogue of anyone of the polypeptide sequences given in Table A4 of Example 1.Preferably, the portion is a portion of a nucleic sequence encoding apolypeptide sequence having in increasing order of preference at least35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99% or more amino acid sequence identity to the IPPT polypeptide asrepresented by SEQ ID NO: 144 or to any of the polypeptide sequencesgiven in Table A4 herein. Most preferably, the portion is a portion ofthe nucleic acid sequence of SEQ ID NO: 143.

Concerning SHR, portions useful in the methods of the invention, encodean SHR polypeptide as defined herein, and have substantially the samebiological activity as the amino acid sequences given in Table A5 ofExample 1. Preferably, the portion is a portion of any one of thenucleic acids given in Table A5 of Example 1, or is a portion of anucleic acid encoding an orthologue or paralogue of any one of the aminoacid sequences given in Table A5 of Example 1. Preferably the portion isat least 1000, 1250, 1500, 1600, 1700 consecutive nucleotides in length,the consecutive nucleotides being of any one of the nucleic acidsequences given in Table A5 of Example 1, or of a nucleic acid encodingan orthologue or paralogue of any one of the amino acid sequences givenin Table A5 of Example 1. Most preferably the portion is a portion ofthe nucleic acid of SEQ ID NO: 208. Preferably, the portion encodes afragment of an amino acid sequence which, when used in the constructionof a GRAS phylogenetic tree, such as the one depicted in FIG. 14,clusters with the group of SHR polypeptides comprising the amino acidsequence represented by SEQ ID NO: 209 rather than with any other group.

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a TCP1 or TCP2, or an Epsin-like, or an IPPT, or a SHRpolypeptide as defined herein, or with a portion as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a nucleic acid capable of hybridizing to any oneof the nucleic acids given in table A of Example 1, or comprisingintroducing and expressing in a plant a nucleic acid capable ofhybridising to a nucleic acid encoding an orthologue, paralogue orhomologue of an amino acid encoded by any of the nucleic acid sequencesgiven in table A of Example 1.

Concerning TCP1 or TCP2 or an Epsine-like-sequences, hybridisingsequences useful in the methods of the invention encode a TCP1 or TCP2or an Epsine-like polypeptide as defined herein, having substantiallythe same biological activity as the amino acid sequences encoded by thenucleic acid sequences given in table A of Example 1. Preferably, thehybridising sequence is capable of hybridising to any one of the nucleicacids given in table A of Example 1, or to a portion of any of thesesequences, a portion being as defined above, or the hybridising sequenceis capable of hybridising to a nucleic acid encoding an orthologue orparalogue of any one of the amino acid sequences encoded by the nucleicacid sequences given in table A of Example 1. Concerning TCP1 or TCP2,most preferably, the hybridising sequence is capable of hybridising to anucleic acid as represented by SEQ ID NO: 1 or to a portion thereof orSEQ ID NO: 3 or to a portion thereof. Concerning Epsine-like sequences,most preferably, the hybridising sequence is capable of hybridising to anucleic acid as represented by SEQ ID NO: 43 or to a portion thereof.

Concerning IPPT, hybridising sequences useful in the methods of theinvention encode an IPPT polypeptide as defined herein, and havesubstantially the same biological activity as the polypeptide sequencesgiven in Table A4 of Example 1. Preferably, the hybridising sequence iscapable of hybridising to any one of the nucleic acid sequences given inTable A4 of Example 1, or to a portion of any of these sequences, aportion being as defined above, or wherein the hybridising sequence iscapable of hybridising to a nucleic acid sequence encoding an orthologueor paralogue of any one of the polypeptide sequences given in Table A4of Example 1. Preferably, the hybridising sequence is capable ofhybridising to a nucleic acid sequence encoding a polypeptide sequencehaving in increasing order of preference at least 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acidsequence identity to the IPPT polypeptide as represented by SEQ ID NO:144 or to any of the polypeptide sequences given in Table A4 herein.Most preferably, the hybridising sequence is capable of hybridising to anucleic acid sequence as represented by SEQ ID NO: 143 or to a portionthereof.

Another nucleic acid sequence variant useful in the methods of theinvention is a splice variant encoding an IPPT polypeptide as definedhereinabove, a splice variant being as defined herein.

Concerning SHR, hybridising sequences useful in the methods of theinvention encode an SHR polypeptide as defined herein, havingsubstantially the same biological activity as the amino acid sequencesgiven in table A of Example 1. Preferably, the hybridising sequence iscapable of hybridising to any one of the nucleic acids given in table Aof Example 1, or to a portion of any of these sequences, a portion beingas defined above, or the hybridising sequence is capable of hybridisingto a nucleic acid encoding an orthologue or paralogue of any one of theamino acid sequences given in table A of Example 1. Most preferably, thehybridising sequence is capable of hybridising to a nucleic acid asrepresented by SEQ ID NO: 208 or to a portion thereof. Preferably, thehybridising sequence encodes a polypeptide with an amino acid sequencewhich, when full-length and used in the construction of a GRASphylogenetic tree, such as the one depicted in FIG. 14, clusters withthe group of SHR polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 209 rather than with any other group.

Concerning TCP1 or TCP2, preferably, the hybridising sequence encodes apolypeptide with an amino acid sequence which, when full-length and usedin the construction of a phylogenetic tree, such as the one depicted inFIG. 1 or 2, clusters with the group of TCP1 or TCP2 polypeptidescomprising the amino acid sequence represented by SEQ ID NO: 2 or 4rather than with any other group.

Concerning Epsin-like-sequences, preferably, the hybridising sequenceencodes a polypeptide with an amino acid sequence which, whenfull-length and used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 3 (Holstein and Oliviusson, Protoplasma 226,13-21, 2005), clusters with the group of Epsin-like polypeptidescomprising the amino acid sequence represented by SEQ ID NO: 44 ratherthan with any other group.

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding a TCP1 or TCP2 or an Epsin-like, or a SHRpolypeptide as defined hereinabove, a splice variant being as definedherein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a splice variant of any one of the nucleic acidsequences given in table A of Example 1, or a splice variant of anucleic acid encoding an orthologue, paralogue or homologue of any ofthe amino acid sequences encoded by the nucleic acid sequences given intable A of Example 1.

Concerning SHR, there is also provided a method for increasing TKW inplants, comprising introducing and expressing in a plant a splicevariant of any one of the nucleic acid sequences given in Table A5 ofExample 1, or a splice variant of a nucleic acid encoding an orthologue,paralogue or homologue of any of the amino acid sequences given in TableA5 of Example 1 and growing the plants under non-nutrient limitingconditions.

Concerning TCP1 or TCP2, preferred splice variants are splice variantsof a nucleic acid represented by SEQ ID NO: 1 or 3, or a splice variantof a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 2 or4. Preferably, the amino acid sequence encoded by the splice variant,when used in the construction of a phylogenetic tree, such as the onedepicted in FIG. 1 or 2, clusters with the group of TCP1 or TCP2polypeptides comprising the amino acid sequence represented by SEQ IDNO: 2 or 4 rather than with any other group.

Concerning Epsin-like sequences, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 43, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 44. Preferably, the amino acid sequence encoded by the splicevariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 3 (Holstein and Oliviusson, Protoplasma 226,13-21, 2005), clusters with the group of Epsin-like polypeptidescomprising the amino acid sequence represented by SEQ ID NO: 44 ratherthan with any other group.

Concerning IPPT, preferred splice variants are splice variants of anucleic acid sequence represented by SEQ ID NO: 143, or a splice variantof a nucleic acid sequence encoding an orthologue or paralogue of SEQ IDNO: 144. Preferably, the splice variant is a splice variant of a nucleicacid sequence encoding a polypeptide sequence having in increasing orderof preference at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to the IPPTpolypeptide as represented by SEQ ID NO: 144 or to any of thepolypeptide sequences given in Table A4 herein.

Concerning SHR, preferred splice variants are splice variants of anucleic acid represented by SEQ ID NO: 208, or a splice variant of anucleic acid encoding an orthologue or paralogue of SEQ ID NO: 209.Preferably, the amino acid sequence encoded by the splice variant, whenused in the construction of a GRAS phylogenetic tree, such as the onedepicted in FIG. 14, clusters with the group of SHR polypeptidescomprising the amino acid sequence represented by SEQ ID NO: 209 ratherthan with any other group.

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding a TCP1 orTCP2 or an Epsin-like, or an IPPT, or an SHR polypeptide as definedhereinabove, an allelic variant being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant an allelic variant of any one of the nucleic acidsgiven in table A of Example 1, or comprising introducing and expressingin a plant an allelic variant of a nucleic acid encoding an orthologue,paralogue or homologue of any of the amino acid sequences encoded by thenucleic acid sequences given in table A of Example 1.

Concerning IPPT, according to the present invention, there is provided amethod for increasing yield-related traits, comprising introducing andexpressing in the seeds of a plant, an allelic variant of any one of thenucleic acid sequences given in table A of Example 1, or comprisingintroducing and expressing in the seeds of a plant, an allelic variantof a nucleic acid sequence encoding an orthologue, paralogue orhomologue of any of the polypeptide sequences given in table A ofExample 1.

Concerning SHR, there is also provided a method for increasing TKW inplants, comprising introducing and expressing in a plant an allelicvariant of any one of the nucleic acids given in Table A of Example 1,or comprising introducing and expressing in a plant an allelic variantof a nucleic acid encoding an orthologue, paralogue or homologue of anyof the amino acid sequences given in Table A of Example 1, and growingplants under non-nutrient limiting conditions.

Concerning TCP1 or TCP2, the allelic variants useful in the methods ofthe present invention have substantially the same biological activity asthe TCP1 or TCP2 polypeptide of SEQ ID NO: 2 or 4 and any of the aminoacids encoded by the nucleic acid sequences given in Example 1. Allelicvariants exist in nature, and encompassed within the methods of thepresent invention is the use of these natural alleles. Preferably, theallelic variant is an allelic variant of SEQ ID NO: 1 or 3 or an allelicvariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 2 or 4. Preferably, the amino acid sequence encoded by the allelicvariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 1 or 2, clusters with the TCP1 or TCP2polypeptides comprising the amino acid sequence represented by SEQ IDNO: 2 or 4 TCP1 or TCP2 rather than with any other group.

Concerning Epsin-like sequences, the allelic variants useful in themethods of the present invention have substantially the same biologicalactivity as the Epsin-like polypeptide of SEQ ID NO: 44 and any of theamino acids depicted in Table A3 of Example 1. Allelic variants exist innature, and encompassed within the methods of the present invention isthe use of these natural alleles. Preferably, the allelic variant is anallelic variant of SEQ ID NO: 43 or an allelic variant of a nucleic acidencoding an orthologue or paralogue of SEQ ID NO: 44. Preferably, theamino acid sequence encoded by the allelic variant, when used in theconstruction of a phylogenetic tree, such as the one depicted in FIG. 3(Holstein and Oliviusson, Protoplasma 226, 13-21, 2005), clusters withthe group of Epsin-like polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 44 rather than with any other group.

Concerning IPPT, the allelic variants useful in the methods of thepresent invention have substantially the same biological activity as theIPPT polypeptide of SEQ ID NO: 144 and any of the polypeptide sequencesdepicted in Table A4 of Example 1. Allelic variants exist in nature, andencompassed within the methods of the present invention is the use ofthese natural alleles. Preferably, the allelic variant is an allelicvariant of SEQ ID NO: 143 or an allelic variant of a nucleic acidsequence encoding an orthologue or paralogue of SEQ ID NO: 144.Preferably, the allelic variant is an allelic variant of a polypeptidesequence having in increasing order of preference at least 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity to the IPPT polypeptide as represented bySEQ ID NO: 144 or to any of the polypeptide sequences given in Table A4herein.

Concerning SHR, the allelic variants useful in the methods of thepresent invention have substantially the same biological activity as theSHR polypeptide of SEQ ID NO: 209 and any of the amino acids depicted inTable A5 of Example 1. Allelic variants exist in nature, and encompassedwithin the methods of the present invention is the use of these naturalalleles. Preferably, the allelic variant is an allelic variant of SEQ IDNO: 208 or an allelic variant of a nucleic acid encoding an orthologueor paralogue of SEQ ID NO: 209. Preferably, the amino acid sequenceencoded by the allelic variant, when used in the construction of a GRASphylogenetic tree, such as the one depicted in FIG. 14, clusters withthe SHR polypeptides comprising the amino acid sequence represented bySEQ ID NO: 209 rather than with any other group.

Gene shuffling or directed evolution may also be used to generatevariants of nucleic acids encoding TCP1 or TCP2 or an Epsin-like, orIPPT, or SHR polypeptides as defined above; the term “gene shuffling”being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related or for increasing TKW traits in plants,comprising introducing and expressing in a plant a variant of any one ofthe nucleic acid sequences given in table A of Example 1, or comprisingintroducing and expressing in a plant a variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences encoded by the nucleic acid sequences given in table A ofExample 1, which variant nucleic acid is obtained by gene shuffling, andgrowing the plants under non-nutrient limiting conditions.

Concerning TCP1 or TCP2, preferably, the amino acid sequence encoded bythe variant nucleic acid obtained by gene shuffling, when used in theconstruction of a phylogenetic tree such as the one depicted in FIG. 1or 2, clusters with the group of TCP1 or TCP2 polypeptides comprisingthe amino acid sequence represented by SEQ ID NO: 2 or 4 rather thanwith any other group.

Concerning Epsin-like sequences, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling, whenused in the construction of a phylogenetic tree such as the one depictedin FIG. 3 (Holstein and Oliviusson, Protoplasma 226, 13-21, 2005),clusters with the group of Epsin-like polypeptides comprising the aminoacid sequence represented by SEQ ID NO: 44 rather than with any othergroup.

Concerning IPPT, preferably, the variant nucleic acid sequence obtainedby gene shuffling encodes a polypeptide sequence having in increasingorder of preference at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identityto the IPPT polypeptide as represented by SEQ ID NO: 144 or to any ofthe polypeptide sequences given in Table A4 herein.

Concerning SHR, preferably, the amino acid sequence encoded by thevariant nucleic acid obtained by gene shuffling, when used in theconstruction of a GRAS phylogenetic tree such as the one depicted inFIG. 14, clusters with the group of SHR polypeptides comprising theamino acid sequence represented by SEQ ID NO: 209 rather than with anyother group.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (Current Protocolsin Molecular Biology. Wiley Eds.).

Nucleic acids encoding TCP1 or TCP2 polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the TCP1 or TCP2 polypeptide-encodingnucleic acid is from a plant, further preferably from a dicotyledonousplant, more preferably from the family Medicago, most preferably thenucleic acid is from Medicago sativa or Medicago truncatula.

Nucleic acids encoding Epsin-like polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the Epsin-like polypeptide-encodingnucleic acid is from a plant, further preferably from a dicotyledonousplant, more preferably from the family Brassicaceae, most preferably thenucleic acid is from Arabidopsis thaliana.

Nucleic acid sequences encoding IPPT polypeptides may be derived fromany natural or artificial source. The nucleic acid sequence may bemodified from its native form in composition and/or genomic environmentthrough deliberate human manipulation. The nucleic acid sequenceencoding an IPPT polypeptide is from the Procaryota domain, preferablyfrom Cyanobacteria, further preferably from the orders Nostocales,Oscillatoriales, Chroococcales, Prochlorales, Gloeobacterales,Pleurocapsales, Stigonematales. More preferably, the nucleic acidsequence encoding an IPPT polypeptide is from Nostoc, Trichodesmium,Anabaena, Acaryochloris, Microcystis, Thermosynechococcus,Synechococcus, Prochiorococcus, Gloeobacter, Synechocystis. Mostpreferably, the nucleic acid sequence encoding an IPPT polypeptide isfrom Synechococcus species, in particular from Synechococcus PCC 7942.

Nucleic acids encoding SHR polypeptides may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the SHR polypeptide-encoding nucleic acid isfrom a plant, further preferably from a dicotyledonous plant, morepreferably from the family Brasicaceae, most preferably the nucleic acidis from Arabidopsis thaliana.

Performance of the methods of the invention gives plants having enhancedyield-related traits. In particular performance of the methods of theinvention gives plants having increased yield, especially increased seedyield relative to control plants. The terms “yield” and “seed yield” aredescribed in more detail in the “definitions” section herein.

Reference herein to enhanced yield-related traits is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground. In particular, such harvestable parts are vegetative plant partsand/or seeds, and performance of the methods of the invention results inplants having increased seed yield relative to the seed yield of controlplants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established persquare meter, an increase in the number of ears per plant, an increasein the number of rows, number of kernels per row, kernel weight,thousand kernel weight, ear length/diameter, increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), among others. Taking rice as anexample, a yield increase may manifest itself as an increase in one ormore of the following: number of plants per square meter, number ofpanicles per plant, number of spikelets per panicle, number of flowers(florets) per panicle (which is expressed as a ratio of the number offilled seeds over the number of primary panicles), increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), increase in thousand kernelweight, among others.

The present invention provides a method for increasing yield, especiallyseed yield of plants, relative to control plants, which method comprisesmodulating expression in a plant of a nucleic acid encoding a TCP1 orTCP2 or an Epsin-like or an IPPT, or an SHR polypeptide as definedherein.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of control plants at a corresponding stage in their lifecycle.

The increased growth rate may be specific to one or more parts of aplant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as early vigour, growth rate, greennessindex, flowering time and speed of seed maturation. The increase ingrowth rate may take place at one or more stages in the life cycle of aplant or during substantially the whole plant life cycle. Increasedgrowth rate during the early stages in the life cycle of a plant mayreflect enhanced vigour. The increase in growth rate may alter theharvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time). If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoybean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per square meter (due to anincrease in the number of times (say in a year) that any particularplant may be grown and harvested). An increase in growth rate may alsoallow for the cultivation of transgenic plants in a wider geographicalarea than their wild-type counterparts, since the territoriallimitations for growing a crop are often determined by adverseenvironmental conditions either at the time of planting (early season)or at the time of harvesting (late season). Such adverse conditions maybe avoided if the harvest cycle is shortened. The growth rate may bedetermined by deriving various parameters from growth curves, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression in a plant of anucleic acid encoding a TCP1 or TCP2 or an Epsin-like, or an IPPTpolypeptide as defined herein. Additionally, a method is provided forincreasing the growth rate of plants, which method comprises modulatingexpression in a plant of a nucleic acid encoding a SHR polypeptide asdefined herein, and growing the plants under conditions of sub-optimalnutrient availability.

Enhanced yield-related traits are obtained by performance of the methodsof the invention and growing plants under conditions of nutrientdeficiency, particularly under conditions of nitrogen deficiency.Nutrient deficiency may result from a lack of nutrients such asnitrogen, phosphates and other phosphorous-containing compounds,potassium, calcium, cadmium, magnesium, manganese, iron and boron,amongst others. According to a preferred feature of the presentinvention, there is provided a method for enhancing yield-related traitsin plants, comprising modulating expression in a plant of a nucleic acidencoding a SHR polypeptide and growing plants under conditions ofnitrogen deficiency.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi, nematodes, and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signalling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Concerning IPPT, since diverse environmental stresses activate similarpathways, the exemplification of the present invention with droughtstress should not be seen as a limitation to drought stress, but more asa screen to indicate the involvement of IPPT polypeptides as definedabove, in increasing yield-related traits relative to control plantsgrown in comparable stress conditions, in abiotic stresses in general.

Concerning IPPT, the term “abiotic stress” as defined herein is taken tomean any one or more of: water stress (due to drought or excess water),anaerobic stress, salt stress, temperature stress (due to hot, cold orfreezing temperatures), chemical toxicity stress and oxidative stress.According to one aspect of the invention, the abiotic stress is anosmotic stress, selected from water stress, salt stress, oxidativestress and ionic stress. Preferably, the water stress is drought stress.The term salt stress is not restricted to common salt (NaCl), but may beany stress caused by one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2,amongst others.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises modulating expression ina plant of a nucleic acid encoding a TCP1 or TCP2 or an Epsin-likepolypeptide.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesmodulating expression in a plant of a nucleic acid encoding a TCP1 orTCP2 or an Epsin-like polypeptide. Nutrient deficiency may result from alack of nutrients such as nitrogen, phosphates and otherphosphorous-containing compounds, potassium, calcium, cadmium,magnesium, manganese, iron and boron, amongst others.

Concerning IPPT, performance of the methods of the invention givesplants having increased yield-related traits, under abiotic stressconditions relative to control plants grown in comparable stressconditions. Therefore, according to the present invention, there isprovided a method for increasing yield-related traits, in plants grownunder abiotic stress conditions, which method comprises increasingexpression in the seeds of a plant, of a nucleic acid sequence encodingan IPPT polypeptide. According to one aspect of the invention, theabiotic stress is an osmotic stress, selected from one or more of thefollowing: water stress, salt stress, oxidative stress and ionic stress.

Furthermore, concerning IPPT, performance of the methods of theinvention gives plants grown under conditions of reduced nutrientavailability, particularly under conditions of reduced nitrogenavailability, having increased yield-related traits relative to controlplants grown under comparable conditions. Therefore, according to thepresent invention, there is provided a method for increasingyield-related traits in plants grown under conditions of reducednutrient availability, preferably reduced nitrogen availability, whichmethod comprises increasing expression in the seeds of a plant, of anucleic acid sequence encoding an IPPT polypeptide. Reduced nutrientavailability may result from a deficiency or excess of nutrients such asnitrogen, phosphates and other phosphorous-containing compounds,potassium, calcium, cadmium, magnesium, manganese, iron and boron,amongst others. Preferably, reduced nutrient availability is reducednitrogen availability.

Another example of abiotic environmental stress is the reducedavailability of one or more nutrients that need to be assimilated by theplants for growth and development. Because of the strong influence ofnutrition utilization efficiency on plant yield and product quality, ahuge amount of fertilizer is poured onto fields to optimize plant growthand quality. Productivity of plants ordinarily is limited by threeprimary nutrients, phosphorous, potassium and nitrogen, which is usuallythe rate-limiting element in plant growth of these three. Therefore themajor nutritional element required for plant growth is nitrogen (N). Itis a constituent of numerous important compounds found in living cells,including amino acids, proteins (enzymes), nucleic acids, andchlorophyll. 1.5% to 2% of plant dry matter is nitrogen andapproximately 16% of total plant protein. Thus, nitrogen availability isa major limiting factor for crop plant growth and production (Frink etal. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well amajor impact on protein accumulation and amino acid composition.Therefore, of great interest are crop plants with increasedyield-related traits, when grown under nitrogen-limiting conditions.

The present invention encompasses plants or parts thereof (includingseeds) or cells thereof obtainable by the methods according to thepresent invention. The plants or parts thereof or cells thereof comprisea nucleic acid transgene encoding a TCP1 or TCP2 or an Epsin-like, or anSHR polypeptide as defined above. Concerning IPPT, the plants or partsthereof or cells thereof comprise a nucleic acid transgene encoding anIPPT polypeptide as defined above, operably linked to a seed-specificpromoter.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression in plants of nucleic acids encoding TCP1or TCP2 or Epsin-like, or IPPT, or SHR polypeptides. The gene constructsmay be inserted into vectors, which may be commercially available,suitable for transforming into plants and suitable for expression of thegene of interest in the transformed cells. The invention also providesuse of a gene construct as defined herein in the methods of theinvention.

More specifically, the present invention provides a constructcomprising:

(a) a nucleic acid encoding a TCP1 or TCP2 or an Epsin-like, or an IPPT,or an SHR polypeptide as defined above;(b) one or more control sequences capable of driving expression of thenucleic acid sequence of (a); and optionally(c) a transcription termination sequence.

Preferably, the nucleic acid encoding a TCP1 or TCP2 or an Epsin-like,or an IPPT, or SHR polypeptide is as defined above. The term “controlsequence” and “termination sequence” are as defined herein.

Plants are transformed with a vector comprising any of the nucleic acidsdescribed above. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells containing the sequence ofinterest. The sequence of interest is operably linked to one or morecontrol sequences (at least to a promoter).

Advantageously, any type of promoter, whether natural or synthetic, maybe used to drive expression of the nucleic acid sequence. A constitutivepromoter is particularly useful in the methods. Preferably theconstitutive promoter is also a ubiquitous promoter. Concerning IPPT, aseed-specific promoter is particularly useful in the methods. Otherorgan-specific promoters, for example for preferred expression inleaves, stems, tubers, meristems, are useful in performing the methodsof the invention. Developmentally-regulated promoters are also useful inperforming the methods of the invention See the “Definitions” sectionherein for definitions of the various promoter types. See the“Definitions” section herein for definitions of the various promotertypes.

It should be clear that the applicability of the present invention isnot restricted to the TCP1 or TCP2 polypeptide-encoding nucleic acidrepresented by SEQ ID NO: 1 or 3, nor is the applicability of theinvention restricted to expression of a TCP1 or TCP2polypeptide-encoding nucleic acid when driven by a constitutivepromoter.

Concerning TCP1 or TCP2, the constitutive promoter is preferably a GOS2promoter, preferably a GOS2 promoter from rice. Further preferably theconstitutive promoter is represented by a nucleic acid sequencesubstantially similar to SEQ ID NO: 5, most preferably the constitutivepromoter is as represented by SEQ ID NO: 5 (See Table 2b in the“Definitions” section herein for further examples of constitutivepromoters). According to another preferred embodiment, the constitutivepromoter is preferably a High Mobility Group Protein (HMGP) promoter.Further preferably the constitutive promoter is represented by a nucleicacid sequence substantially similar to SEQ ID NO: 6, most preferably theconstitutive promoter is as represented by SEQ ID NO: 6.

It should also be clear that the applicability of the present inventionis not restricted to the Epsin-like polypeptide-encoding nucleic acidrepresented by SEQ ID NO: 43, nor is the applicability of the inventionrestricted to expression of an Epsin-like polypeptide-encoding nucleicacid when driven by a constitutive promoter.

Furthermore, it should be clear that the applicability of the presentinvention is not restricted to a nucleic acid sequence encoding the IPPTpolypeptide, as represented by SEQ ID NO: 143, nor is the applicabilityof the invention restricted to expression of an IPPTpolypeptide-encoding nucleic acid sequence when driven by aseed-specific promoter.

Also, it should be clear that the applicability of the present inventionis not restricted to the SHR polypeptide-encoding nucleic acidrepresented by SEQ ID NO: 208, nor is the applicability of the inventionrestricted to expression of a SHR polypeptide-encoding nucleic acid whendriven by a constitutive promoter.

Concerning Epsin-like sequences, the constitutive promoter is preferablya GOS2 promoter, preferably a GOS2 promoter from rice. Furtherpreferably the constitutive promoter is represented by a nucleic acidsequence substantially similar to SEQ ID NO: 45, most preferably theconstitutive promoter is as represented by SEQ ID NO: 45. See Table 2 inthe “Definitions” section herein for further examples of constitutivepromoters.

Concerning IPPT, preferably, one of the control sequences of a constructis a seed-specific promoter. An example of a seed-specific promoter is adehydrin promoter, preferably a rice dehydrin promoter, more preferablya dehydrin promoter as represented by SEQ ID NO: 204. Alternatively, theseed-specific promoter is a proteinase inhibitor promoter, preferably arice proteinase inhibitor promoter, more preferably a proteinaseinhibitor promoter as represented by SEQ ID NO: 205.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Thoseskilled in the art will be aware of terminator and enhancer sequencesthat may be suitable for use in performing the invention. An intronsequence may also be added to the 5′ untranslated region (UTR) or in thecoding sequence to increase the amount of the mature message thataccumulates in the cytosol, as described in the definitions section.Other control sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements. Such sequences would be known or may readily beobtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene. Selectable markers aredescribed in more detail in the “definitions” section herein. The markergenes may be removed or excised from the transgenic cell once they areno longer needed. Techniques for marker removal are known in the art,useful techniques are described above in the definitions section.

The invention also provides a method for the production of transgenicplants having enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding a TCP1 or TCP2 or an Epsin-like polypeptide as definedhereinabove.

The invention also provides a method for the production of transgenicplants having enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding an SHR polypeptide as defined hereinabove and growing theplants under conditions of sub-optimal nutrient availability.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased enhanced yield-relatedtraits, particularly increased (seed) yield, which method comprises:

(i) introducing and expressing in a plant or plant cell a TCP1 or TCP2polypeptide-encoding nucleic acid; and(ii) cultivating the plant cell under conditions promoting plant growthand development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a TCP1 or TCP2 polypeptide as defined herein.

Concerning Epsin-like sequences, the present invention provides a methodfor the production of transgenic plants having increased enhancedyield-related traits, particularly increased biomass and/or increasedseed yield, which method comprises:

(i) introducing and expressing in a plant or plant cell an Epsin-likepolypeptide-encoding nucleic acid; and(ii) cultivating the plant cell under conditions promoting plant growthand development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding an Epsin-like polypeptide as defined herein.

In another embodiment, the invention provides a method for theproduction of transgenic plants having increased yield-related traitsrelative to control plants, comprising introduction and expression inthe seeds of a plant, of any nucleic acid sequence encoding an IPPTpolypeptide as defined hereinabove.

Concerning IPPT, more specifically, the present invention provides amethod for the production of transgenic plants having increasedyield-related traits relative to control plants, which method comprises:

-   -   (i) introducing and expressing in a plant, plant part, or plant        cell a nucleic acid sequence encoding an IPPT polypeptide, under        the control of seed-specific promoter; and    -   (ii) cultivating the plant cell, plant part or plant under        conditions promoting plant growth and development.

The nucleic acid sequence of (i) may be any of the nucleic acidsequences capable of encoding an IPPT polypeptide as defined herein.

In yet another embodiment, the present invention provides a method forthe production of transgenic plants having enhanced yield-relatedtraits, particularly increased (seed) yield, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell an SHR        polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under conditions of sub-optimal        nutrient availability.

Concerning SHR, the invention also provides a method for the productionof transgenic plants having increased TKW relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding an SHR polypeptide as defined hereinabove, and growing theplants under non-nutrient limiting conditions. More specifically, thepresent invention provides a method for the production of transgenicplants having increased TKW, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell an SHR        polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under non-nutrient limiting        conditions.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding an SHR polypeptide as defined herein.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation. The term “transformation” is described in more detail inthe “definitions” section herein.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a TCP1 or TCP2, or an Epsin-like, or an SHR polypeptide asdefined hereinabove. Preferred host cells according to the invention areplant cells. Concerning IPPT, the invention also includes host cellscontaining an isolated nucleic acid sequence encoding an IPPTpolypeptide as defined hereinabove, operably linked to a seed-specificpromoter. Host plants for the nucleic acids or the vector used in themethod according to the invention, the expression cassette or constructor vector are, in principle, advantageously all plants, which arecapable of synthesizing the polypeptides used in the inventive method.

The methods of the invention are advantageously applicable to any plant.Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes,tubers and bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

Concerning IPPT, the invention also extends to harvestable parts of aplant comprising an isolated nucleic acid sequence encoding an IPPT (asdefined hereinabove) operably linked to a seed-specific promoter, suchas, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes,tubers and bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins. Methods for increasing expression of nucleic acid sequences orgenes, or gene products, are well documented in the art and examples areprovided in the definitions section.

According to a preferred feature of the invention, the modulatedexpression is increased expression. Methods for increasing expression ofnucleic acids or genes, or gene products, are well documented in the artand examples are provided in the definitions section.

As mentioned above, a preferred method for modulating expression of anucleic acid encoding a TCP1 or TCP2 or an Epsin-like, or an SHRpolypeptide is by introducing and expressing in a plant a nucleic acidencoding a TCP1 or TCP2 or an Epsin-like, or an SHR polypeptide; or apreferred method for increasing expression of a nucleic acid sequenceencoding an IPPT polypeptide is by introducing and expressing in theseeds of a plant, a nucleic acid sequence encoding an IPPT polypeptide;however the effects of performing the method, i.e. enhancingyield-related traits may also be achieved using other well knowntechniques, including but not limited to T-DNA activation tagging,TILLING, homologous recombination. A description of these techniques isprovided in the definitions section.

The present invention also encompasses use of nucleic acids encodingTCP1 or TCP2 or an Epsin-like, or an SHR polypeptides as describedherein and use of these TCP1 or TCP2 or an Epsin-like, or an SHRpolypeptides in enhancing any of the aforementioned yield-related traitsin plants. The present invention also encompasses use of nucleic acidsequences encoding IPPT polypeptides as described herein and use ofthese IPPT polypeptides in increasing any of the aforementionedyield-related traits in plants, under normal growth conditions, underabiotic stress growth (preferably osmotic stress growth conditions)conditions, and under growth conditions of reduced nutrientavailability, preferably under conditions of reduced nitrogenavailability.

Nucleic acids encoding TCP1 or TCP2 or an Epsin-like, or an IPPT, or anSHR polypeptide described herein, or the TCP1 or TCP2 or the Epsin-like,or the IPPT, or an SHR polypeptides themselves, may find use in breedingprogrammes in which a DNA marker is identified which may be geneticallylinked to a TCP1 or TCP2 or an Epsin-like, or an IPPT, or an SHRpolypeptide-encoding gene. The nucleic acids/genes, or the TCP1 or TCP2or the Epsin-like, or the IPPT, or an SHR polypeptides themselves may beused to define a molecular marker. This DNA or protein marker may thenbe used in breeding programmes to select plants having enhancedyield-related traits, or increased TKW as defined hereinabove in themethods of the invention.

Allelic variants of a TCP1 or TCP2 or an Epsin-like, or an SHRpolypeptide-encoding nucleic acid/gene may also find use inmarker-assisted breeding programmes. Such breeding programmes sometimesrequire introduction of allelic variation by mutagenic treatment of theplants, using for example EMS mutagenesis; alternatively, the programmemay start with a collection of allelic variants of so called “natural”origin caused unintentionally. Identification of allelic variants thentakes place, for example, by PCR. This is followed by a step forselection of superior allelic variants of the sequence in question andwhich give increased yield. Selection is typically carried out bymonitoring growth performance of plants containing different allelicvariants of the sequence in question. Growth performance may bemonitored in a greenhouse or in the field. Further optional stepsinclude crossing plants in which the superior allelic variant wasidentified with another plant. This could be used, for example, to makea combination of interesting phenotypic features.

Allelic variants of a gene/nucleic acid sequence encoding an IPPTpolypeptide may also find use in marker-assisted breeding programmes.Such breeding programmes sometimes require introduction of allelicvariation by mutagenic treatment of the plants, using for example EMSmutagenesis; alternatively, the programme may start with a collection ofallelic variants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increasedyield-related traits. Selection is typically carried out by monitoringgrowth performance of plants containing different allelic variants ofthe sequence in question. Growth performance may be monitored in agreenhouse or in the field. Further optional steps include crossingplants in which the superior allelic variant was identified with anotherplant. This could be used, for example, to make a combination ofinteresting phenotypic features.

Nucleic acids encoding TCP1 or TCP2 or Epsin-like, or an IPPT, or an SHRpolypeptides may also be used as probes for genetically and physicallymapping the genes that they are a part of, and as markers for traitslinked to those genes. Such information may be useful in plant breedingin order to develop lines with desired phenotypes. Such use of TCP1 orTCP2 or an Epsin-like, or an IPPT, or an SHR polypeptide-encodingnucleic acids requires only a nucleic acid sequence of at least 15nucleotides in length. The TCP1 or TCP2 or an IPPT, or the Epsin-like,or an SHR polypeptide-encoding nucleic acids may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Sambrook J,Fritsch E F and Maniatis T (1989) Molecular Cloning, A LaboratoryManual) of restriction-digested plant genomic DNA may be probed with theTCP1 or TCP2 or the Epsin-like, or an IPPT, or an SHR-encoding nucleicacids. The resulting banding patterns may then be subjected to geneticanalyses using computer programs such as MapMaker (Lander et al. (1987)Genomics 1: 174-181) in order to construct a genetic map. In addition,the nucleic acids may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe TCP1 or TCP2 or the Epsin-like, or an IPPT, or an SHRpolypeptide-encoding nucleic acid in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield-enhancing traits, tolerance to other abiotic and bioticstresses, tolerance to herbicides, insectides, traits modifying variousarchitectural features and/or biochemical and/or physiological features.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 is a multiple alignment of TCP1 polypeptides with the TCP domainand Domain A, B and C boxed.

FIG. 2 is a multiple alignment of TCP2 polypeptides with the TCP domainand Domains 1, 2, 3, 4 and 5 boxed.

FIG. 3 represents the domain structure of SEQ ID NO: 44 with the ENTHdomain as identified in SMART indicated in bold and the conserved motifs1 to 5 underlined.

FIG. 4 represents a multiple alignment of various Epsin-like proteinsequences. The database accession numbers are used as identifiers

FIG. 5 shows a phylogenetic tree of eukaryotic proteins comprising anENTH or ANTH domain (Holstein and Oliviusson 2005). The amino-terminalpart of the proteins (200 amino acids) were aligned using ClustalW 1.82and the output was used in DrawTree (PHYLIP package). SEQ ID NO: 44clusters in the group of the plant ENTHs.

FIG. 6 represents the binary vector for increased expression in Oryzasativa of an Epsin-like-encoding nucleic acid under the control of arice GOS2 promoter (pGOS2)

FIG. 7 details examples of sequences useful in performing the methodsaccording to the present invention.

FIG. 8 schematically represents the two major cytokinin biosyntheticroutes: (a) the adenylate-IPT route, using AMP, ADP, or ATP, and DMAPPor HMBDP, and (b) the tRNA-IPT route using tRNA and DMAPP. According toYevdakova and von Schwartzenberg (2007) Planta 226:683-695.

FIG. 9 shows a detailed model of isoprenoid cytokinin biosynthesispathways, according to Sakakibara (2006) Annu Rev Plant Biol 57.431-449. The tRNA-IPT is indicated by a black arrow in the top rightcorner, the specific final cytokinin product (cZ) of that route is alsoindicated by a black arrow in the bottom right corner.

FIG. 10 shows an AlignX (from Vector NTI 10.3, Invitrogen Corporation)multiple sequence alignment of the IPPT polypeptides from Table A4. TheN-terminal ATP/GTP-binding site motif A (P-loop) as represented by SEQID NO: 199, the Conserved motif I DSR(Q/L)(V/L/I) as represented by SEQID NO: 200, the Conserved motif II (N/D/S/T)(I/V)GTAKP(T/S) asrepresented by SEQ ID NO: 201, the Conserved motif IIIL(V/A/I)GG(S/T)GLY as represented by SEQ ID NO:202, and the Conservedmotif IV F/Y/L)AK(R/K/Q)Q(R/K/M)TWFR, are boxed. The putative zincfinger motif C2H2 (C-X2-C-X(12,18)-H-X5-H found in eukaryotic tRNA-IPTsis marked with a bracket, and the conserved Cys and His residues thereinare boxed.

FIG. 11 shows the binary vector for increased expression in the seeds ofOryza sativa of a nucleic acid sequence encoding an IPPT polypeptideunder the control of either a dehydrin seed-specific promoter, or of aproteinase inhibitor seed-specific promoter from rice.

FIG. 12 details examples of sequences useful in performing the methodsaccording to the present invention.

FIG. 13 shows the structure of GRAS proteins with the 5 motifs typicalto this family

FIG. 14: Neighbour-joining tree of GRAS and SHR proteins. GRAS proteinsfrom rice, Arabidopsis and SHR-related proteins from the variousorganisms were aligned using MUSCLE. A neighbour-joining tree wasproduced with CLUSTALX. Bootstrap analysis was performed for 100iterations. The bootstrap support is shown only for the main nodes. TheSHR related proteins are indicated. A. thaliana: Arabidopsis thaliana;E. grandis: Eucalyptus grandis; G. max: Glycine max; L. sativa: Latucasativa; M. trucatula: Medicago truncatula; O. sativa: Oryza sativa; P.taeda: Pinus taeda; P. patens: Physcomitrella patens; P. trichocarpa:Populus trichocarpa; R. communis: Ricinus communis; S. tuberosum:Solanum tuberosum; V. vinifera: Vitis vinifera; Z. mays: Zea mays;—part:partial sequence.

FIG. 15 shows the percentage sequence identity for members of the GRASfamily with entries above the horizontal line indicating members of theSHR family. The SHR branch is highly conserved in land plants, includingmosses and gymnosperms. The SHR proteins in that branch share more than41% identity with each other, compared with less than 33% with themembers of the other branches.

FIG. 16 represents the binary vector for increased expression in Oryzasativa of a SHR-encoding nucleic acid under the control of a rice GOS2promoter (pGOS2)

FIG. 17 details examples of sequences useful in performing the methodsaccording to the present invention.

In one embodiment the invention relates to subject mater summarized asfollows:

Item 1: Method for enhancing yield-related traits in plants relative tocontrol plants, comprising modulating expression of a nucleic acidencoding a TCP1 or a TCP2 polypeptide in a plant, said TCP1 polypeptidecomprising:

(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of any one of the sequences indicated in FIG. 1; and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain A of any one of the sequences indicated in FIG. 1; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain B of any one of the sequences indicated in FIG. 1; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain C of any one of the sequences indicated in FIG. 1,and said TCP2 polypeptide comprising:(i) a TCP domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe TCP domain of any of the sequences indicated in FIG. 2; and(ii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 1 of any of the sequences indicated in FIG. 2; and(iii) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 2 of any of the sequences indicated in FIG. 2; and(iv) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 3 of any of the sequences indicated in FIG. 2.

Item 2: Method according to Item 1, wherein said TCP2 polypeptidecomprises:

(v) a domain having in increasing order of preference at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 4 of any of the sequences indicated in FIG. 2;(vi) a domain having in increasing order of preference at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 5 of any of the sequences indicated in FIG. 2.

Item 3: Method according to Item 1 or 2, wherein said TCP1 polypeptide,when used in the construction of a TCP phylogenetic tree, such as theone depicted in FIG. 1, tends to cluster with the Glade of TCPpolypeptides comprising the polypeptide sequence as represented by SEQID NO: 2 rather than with any other TCP Glade.

Item 4: Method according to Item 1 or 2, wherein said TCP2 polypeptide,when used in the construction of a TCP phylogenetic tree, such as theone depicted in FIG. 2, tends to cluster with the Glade of TCPpolypeptides comprising the polypeptide sequence as represented by SEQID NO: 4 rather than with any other TCP Glade.

Item 5: Method according to any one of the preceding Items, wherein saidnucleic acid sequence encodes an orthologue or paralogue of SEQ ID NO: 2or 4.

Item 6: Method according to any one of the preceding Items, wherein saidmodulated expression is increased expression of a nucleic acid encodinga TCP1 or a TCP2 polypeptide.

Item 7: Method according to Item 6, wherein said increased expression iseffected by any one or more of T-DNA activation tagging, TILLING, orhomologous recombination.

Item 8: Method according to Item 6, wherein said increased expression iseffected by introducing and expressing in a plant a nucleic acidsequence encoding a TCP1 or a TCP2 polypeptide.

Item 9: Method according to any one of the preceding Items, wherein saidenhanced yield-related traits comprise increased seed weight relative tocontrol plants.

Item 10: Method according to Items 8 or 9, wherein said nucleic acidsequence is operably linked to a constitutive promoter, preferably to aHMGP (High Mobility Group Protein) promoter or to a GOS2 promoter.

Item 11: Method according to any one of Items 7 to 9, wherein saidnucleic acid sequence encoding a TCP1 or TCP2 polypeptide is preferablyof plant origin, further preferably from a dicotyledonous plant, morepreferably from the Medicago family, most preferably from Medicagosativa or Medicago truncatula.

Item 12: Plant or part thereof including seeds obtainable by a methodaccording to any one of Items 1 to 11, wherein said plant or partthereof comprises a nucleic acid transgene encoding a TCP1 or a TCP2polypeptide.

Item 13: Construct comprising:

(i) nucleic acid sequence encoding a TCP1 or a TCP2 polypeptide;(ii) one or more control sequences capable of driving expression of thenucleic acid sequence of (i); and optionally(iii) a transcription termination sequence.

Item 14: Construct according to Item 13, wherein said one or morecontrol sequences is at least a constitutive promoter, preferably anHMGP or GOS2 promoter.

Item 15: Use of a construct according to Items 13 or 14 for makingplants having increased yield, particularly seed yield, relative tocontrol plants.

Item 16: Plant, plant part, or plant cell transformed with a constructaccording to Items 13 or 14.

Item 17: Method for the production of a transgenic plant havingincreased seed yield relative to control plants, which method comprises:

(i) introducing and expressing in a plant or plant cell a nucleic acidsequence encoding a TCP1 or a TCP2 polypeptide; and(ii) cultivating the plant cell under conditions promoting plant growthand development.

Item 18: Transgenic plant having increased yield, particularly increasedseed yield, relative to control plants, said increased yield resultingfrom increased expression of a nucleic acid encoding a TCP1 or a TCP2polypeptide, or a transgenic plant cell derived from said transgenicplant.

Item 19: Transgenic plant according to Item 18, wherein said increasedseed yield is one or more of the following: (i) increased seed weight;(ii) increased harvest index; or (iii) increased Thousand Kernel Weight,(iv) increased number of flowers per panicle, (v) increased fill rate,(vi) increased number of filled seeds.

Item 20: Transgenic plant according to Item 12, 16, 18 or 19, whereinsaid plant is a crop plant or a monocot or a cereal, such as rice,maize, wheat, barley, millet, rye, sorghum and oats, or a transgenicplant cell derived from said transgenic plant.

Item 21: Harvestable parts of a plant according to Item 20, wherein saidharvestable parts are preferably seeds.

Item 22: Products derived from a plant according to Item 20 and/or fromharvestable parts of a plant according to Item 21.

Item 23: Use of a nucleic acid encoding a TCP1 or TCP2 polypeptide inincreasing yield, particularly seed yield in plants.

Item 24: A method for enhancing yield-related traits in plants relativeto control plants, comprising modulating expression in a plant of anucleic acid encoding an Epsin-like polypeptide, wherein said Epsin-likepolypeptide comprises an ENTH domain.

Item 25: Method according to Item 24, wherein said Epsin-likepolypeptide comprises two or more of the following motifs:

(i)Motif 1: (SEQ ID NO: 48)(V/I)(L/R)(D/E)AT(S/D/N)(N/D/E/S)E(P/S)WGPHG(T/S/E), (ii)Motif 2:(SEQ ID NO: 49)F(Q/E)(Y/F)(I/L/V/R/K)(D/E)(S/P/A)(S/G/N/Q/R)G(R/K)D(Q/V/A/H/E)G(S/N/L/I/V)NVR,(iii)Motif 3: (SEQ ID NO: 50)(E/S/A/Q)(V/I/E/A)R(Q/E/D/N)KA(A/L/V/E)(A/V/S/R/K)(N/T)(R/A)(D/E/N/G)K(iv)Motif 4: (SEQ ID NO: 51) WAD(T/S)LSRGL(V/I) (v)Motif 5:(SEQ ID NO: 52) L(A/S)D(I/V)G(I/V)(D/V)(F/G)(D/E/P/G)

Item 26: Method according to Item 24 or 25, wherein said modulatedexpression is effected by introducing and expressing in a plant anucleic acid encoding an Epsin-like polypeptide.

Item 27: Method according to any preceding Item 24 to 26, wherein saidnucleic acid encoding an Epsin-like polypeptide encodes any one of theproteins listed in Table A or is a portion of such a nucleic acid, or anucleic acid capable of hybridising with such a nucleic acid.

Item 28: Method according to any preceding Item 24 to 27, wherein saidnucleic acid sequence encodes an orthologue or paralogue of any of theproteins given in Table A.

Item 29: Method according to any preceding Item 24 to 28, wherein saidenhanced yield-related traits comprise increased yield, preferablyincreased biomass and/or increased seed yield relative to controlplants.

Item 30: Method according to any one of Items 24 to 29, wherein saidenhanced yield-related traits are obtained under non-stress conditions.

Item 31: Method according to any one of Items 24 to 29, wherein saidenhanced yield-related traits are obtained under conditions of milddrought.

Item 32: Method according to any one of Items 26 to 31, wherein saidnucleic acid is operably linked to a constitutive promoter, preferablyto a GOS2 promoter, most preferably to a GOS2 promoter from rice.

Item 33: Method according to any preceding Item 24 to 32, wherein saidnucleic acid encoding an Epsin-like polypeptide is of plant origin,preferably from a dicotyledonous plant, further preferably from thefamily Brassicaceae, more preferably from the genus Arabidopsis, mostpreferably from Arabidopsis thaliana.

Item 34: Plant or part thereof, including seeds, obtainable by a methodaccording to any preceding Item 24 to 33, wherein said plant or partthereof comprises a recombinant nucleic acid encoding an Epsin-likepolypeptide.

Item 35: Construct comprising:

(i) nucleic acid encoding an Epsin-like polypeptide as defined in Items24 or 25;(ii) one or more control sequences capable of driving expression of thenucleic acid sequence of (a); and optionally(iii) a transcription termination sequence.

Item 36: Construct according to Item 35, wherein one of said controlsequences is a constitutive promoter, preferably a GOS2 promoter, mostpreferably a GOS2 promoter from rice.

Item 37: Use of a construct according to Item 35 or 36 in a method formaking plants having increased yield, particularly increased seed yieldrelative to control plants.

Item 38: Plant, plant part or plant cell transformed with a constructaccording to Item 35 or 36.

Item 39: Method for the production of a transgenic plant havingincreased yield, particularly increased biomass and/or increased seedyield relative to control plants, comprising:

(i) introducing and expressing in a plant a nucleic acid encoding anEpsin-like polypeptide as defined in Item 24 or 25; and(ii) cultivating the plant cell under conditions promoting plant growthand development.

Item 40: Transgenic plant having increased yield, particularly increasedbiomass and/or increased seed yield, relative to control plants,resulting from modulated expression of a nucleic acid encoding anEpsin-like polypeptide as defined in Item 24 or 25, or a transgenicplant cell derived from said transgenic plant.

Item 41: Transgenic plant according to Item 34, 38 or 40, or atransgenic plant cell derived thereof, wherein said plant is a cropplant or a monocot or a cereal, such as rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

Item 42: Harvestable parts of a plant according to Item 41, wherein saidharvestable parts are seeds.

Item 43: Products derived from a plant according to Item 41 and/or fromharvestable parts of a plant according to Item 42.

Item 44: Use of a nucleic acid encoding an Epsin-like polypeptide inincreasing yield, particularly in increasing seed yield in plants,relative to control plants.

Item 45: An isolated nucleic acid molecule comprising a nucleic acidmolecule selected from the group consisting of:

a) a nucleic acid molecule encoding the polypeptide shown in SEQ IDNO:112, SEQ ID NO:138 and SEQ ID NO:142;b) a nucleic acid molecule shown in SEQ ID NO:111, SEQ ID NO:137 and SEQID NO:141;c) a nucleic acid molecule, which, as a result of the degeneracy of thegenetic code, can be derived from a polypeptide sequence depicted in SEQID NO:112, SEQ ID NO:138 and SEQ ID NO:142 and confers enhancedyield-related traits in plants relative to control plants;d) a nucleic acid molecule having, in increasing order of preference, atleast 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one ofthe amino acid sequences given in SEQ ID NO:111, SEQ ID NO:137 and SEQID NO:141 and confers enhanced yield-related traits in plants relativeto control plants;e) a nucleic acid molecule encoding a polypeptide, in increasing orderof preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequenceidentity with the amino acid sequence of the polypeptide encoded by thenucleic acid molecule of (a) to (c) and confers enhanced yield-relatedtraits in plants relative to control plants;f) nucleic acid molecule which hybridizes with a nucleic acid moleculeof (a) to (c) under stringent hybridization conditions and confersenhanced yield-related traits in plants relative to control plants;g) a nucleic acid molecule encoding a polypeptide which can be isolatedwith the aid of monoclonal or polyclonal antibodies made against apolypeptide encoded by one of the nucleic acid molecules of (a) to (e);h) a nucleic acid molecule encoding a polypeptide comprising theconsensus sequence or one or more polypeptide motifs as shown in Motif 1(corresponding to SEQ ID NO: 6), Motif 2 (corresponding to SEQ ID NO:7), Motif 3 (corresponding to SEQ ID NO: 8), Motif 4 (corresponding toSEQ ID NO: 9) or Motif 5 (corresponding to SEQ ID NO: 10);i) nucleic acid molecule which comprises a polynucleotide, which isobtained by amplifying a cDNA library or a genomic library using theprimers shown in SEQ ID NO: 46 (prm09481) and SEQ ID NO: 47 (prm09482)andj) a nucleic acid molecule which is obtainable by screening a suitablenucleic acid library under stringent hybridization conditions with aprobe comprising a complementary sequence of a nucleic acid molecule of(a) or (b) or with a fragment thereof, having at least 15 nt, preferably20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid moleculecomplementary to a nucleic acid molecule sequence characterized in (a)to (e).

Item 46: Polypeptide encoded by a nucleic acid molecule according toitem 45.

Item 47: A method for increasing yield-related traits in plants relativeto control plants, comprising increasing expression in the seeds of aplant, of a nucleic acid sequence encoding a tRNAdelta(2)-isopentenylpyrophosphate transferase (IPPT) polypeptide, whichIPPT polypeptide comprises (i) a tRNA isopentenyltransferase domain withan InterPro accession IPR002627; and (ii) an N-terminal ATP/GTP-bindingsite motif A (P-loop), and optionally selecting for plants havingincreased yield-related traits.

Item 48: Method according to Item 47, wherein said IPPT polypeptide has(i) in increasing order of preference at least 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% or more amino acid sequence identity to an N-terminalATP/GTP-binding site motif A (P-loop) as represented by SEQ ID NO: 199;and has in increasing order of preference at least 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or more amino acid sequence identity to one or moreof: (ii) Conserved motif I DSR(Q/L)(V/L/I) as represented by SEQ ID NO:200; or (ii) Conserved motif II (N/D/S/T)(I/V)GTAKP(T/S) as representedby SEQ ID NO: 201; or (iii) Conserved motif III L(V/A/I)GG(S/T)GLY asrepresented by SEQ ID NO:202; or (iv) Conserved motif IVF/Y/L)AK(R/K/Q)Q(R/K/M)TWFR as represented by SEQ ID NO: 203.

Item 49: Method according to Item 47 or 48, wherein said IPPTpolypeptide has in increasing order of preference at least 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity to the IPPT polypeptide as represented bySEQ ID NO: 144 or to any of the polypeptide sequences given in Table Aherein.

Item 50: Method according to any preceding Item 47 to 49, wherein saidIPPT polypeptide is capable of complementing a yeast mod 5 mutant strainwhich lacks endogenous IPPT activity, or is capable of complementing anE. coli miaA mutant strain which lacks endogenous IPPT activity,

Item 51: Method according to any preceding Item 47 to 50, wherein saidnucleic acid sequence encoding an IPPT polypeptide is represented by anyone of the nucleic acid sequence SEQ ID NOs given in Table A or aportion thereof, or a sequence capable of hybridising with any one ofthe nucleic acid sequences SEQ ID NOs given in Table A.

Item 52: Method according to any preceding Item 47 to 51, wherein saidnucleic acid sequence encodes an orthologue or paralogue of any of thepolypeptide sequence SEQ ID NOs given in Table A.

Item 53: Method according to any preceding Item 47 to 52, wherein saidincreased expression is effected by any one or more of: T-DNA activationtagging, TILLING, or homologous recombination.

Item 54: Method according to any preceding Item 47 to 53, wherein saidincreased expression is effected by introducing and expressing in theseeds of a plant, a nucleic acid sequence encoding an IPPT polypeptide.

Item 55: Method according to any preceding Item 47 to 54, wherein saidincreased yield-related trait is one or more of: increased early vigour,increased aboveground biomass, increased total seed yield per plant,increased total number of seeds, increased number of filled seeds,increased number of flowers per panicles, and increased harvest index.

Item 56: Method according to any preceding Item 47 to 55, wherein saidnucleic acid sequence is operably linked to a seed-specific promoter.

Item 57: Method according to Item 56, wherein said seed-specificpromoter is a dehydrin promoter, preferably a rice dehydrin promoter,more preferably a dehydrin promoter as represented by SEQ ID NO: 204.

Item 58: Method according to Item 56, wherein said seed-specificpromoter is a proteinase inhibitor promoter, preferably to a riceproteinase inhibitor promoter, more preferably a proteinase inhibitorpromoter as represented by SEQ ID NO: 205.

Item 59: Method according to any preceding Item 47 to 58 wherein saidnucleic acid sequence encoding an IPPT polypeptide is from theProcaryota domain, preferably from Cyanobacteria, further preferablyfrom Chroococcales, more preferably from Synechococcus species, mostpreferably from Synechococcus PCC 7942.

Item 60: Plants, parts thereof (including seeds), or plant cellsobtainable by a method according to any preceding Item 47 to 59, whereinsaid plant, part or cell thereof comprises an isolated nucleic acidtransgene encoding an IPPT polypeptide, operably linked to aseed-specific promoter.

Item 61: Construct comprising:

(a) a nucleic acid sequence encoding an IPPT polypeptide as defined inany one of Items 47 to 52;(b) one or more control sequences capable of driving expression of thenucleic acid sequence of (a); and optionally(c) a transcription termination sequence.

Item 62: Construct according to Item 61, wherein said control sequenceis a seed-specific promoter.

Item 63: Construct according to Item 62, wherein said seed-specificpromoter is a dehydrin promoter, preferably a rice dehydrin promoter,more preferably a dehydrin promoter as represented by SEQ ID NO: 204.

Item 64: Construct according to Item 62, wherein said seed-specificpromoter is a proteinase inhibitor promoter, preferably to a riceproteinase inhibitor promoter, more preferably a proteinase inhibitorpromoter as represented by SEQ ID NO: 205.

Item 65: Use of a construct according to any one of Items 61 to 64, in amethod for making plants having increased yield-related traits relativeto control plants, which increased yield-related traits are one or moreof: increased early vigour, increased aboveground biomass, increasedtotal seed yield per plant, increased total number of seeds, increasednumber of filled seeds, increased number of flowers per panicles, andincreased harvest index.

Item 66: Plant, plant part or plant cell transformed with a constructaccording to any one of Items 61 to 64.

Item 67: Method for the production of transgenic plants having increasedyield-related traits relative to control plants, comprising:

(i) introducing and expressing in a plant, plant part, or plant cell, anucleic acid sequence encoding an IPPT polypeptide as defined in any oneof Items 47 to 52, under the control of a seed-specific promoter; and(ii) cultivating the plant cell, plant part, or plant under conditionspromoting plant growth and development.

Item 68: Transgenic plant having increased yield-related traits relativeto control plants, resulting from increased expression in the seeds, ofa nucleic acid sequence encoding an IPPT polypeptide as defined in anyone of Items 47 to 52, operably linked to a seed-specific promoter, or atransgenic plant cell or transgenic plant part derived from saidtransgenic plant.

Item 69: Transgenic plant according to Item 60, 66 or 68, wherein saidplant is a crop plant or a monocot or a cereal, such as rice, maize,wheat, barley, millet, rye, triticale, sorghum and oats, or a transgenicplant cell derived from said transgenic plant.

Item 70: Harvestable parts comprising an isolated nucleic acid sequenceencoding an IPPT polypeptide of a plant according to Item 69, whereinsaid harvestable parts are preferably seeds.

Item 71: Products derived from a plant according to Item 69 and/or fromharvestable parts of a plant according to Item 70.

Item 72: Use of a nucleic acid sequence encoding an IPPT polypeptide asdefined in any one of Items 45 to 50 in increasing yield-related traits,comprising one or more of increased early vigour, increased abovegroundbiomass, increased total seed yield per plant, increased total number ofseeds, increased number of filled seeds, increased number of flowers perpanicles, and increased harvest index.

Item 73: A method for enhancing yield related traits in plants relativeto control plants, comprising modulating expression of a nucleic acidencoding an SHR polypeptide in plants grown under conditions ofsub-optimal nutrient availability.

Item 74: A method for increasing Thousand Kernel Weight (TKW) in plantsrelative to control plants, comprising modulating expression of anucleic acid encoding an SHR polypeptide in plants grown undernon-nutrient limiting conditions.

Item 75: Method according to Item 73 or 74, wherein said SHR polypeptidecomprises any full length polypeptide which when used in theconstruction of a GRAS phylogenetic tree, such as the one depicted inFIG. 2, clusters with the group of SHR polypeptides comprising the aminoacid sequence represented by SEQ ID NO: 209 rather than with any othergroup.

Item 76: Method according to Item 73 or 75, wherein said conditions ofsub-optimal nutrient availability are reduced nitrogen availabilityrelative to control plants.

Item 77: Method according to any preceding Item 73 to 76, wherein saidmodulated expression is effected by introducing and expressing in aplant a nucleic acid encoding an SHR polypeptide.

Item 78: Method according to any preceding Item 73 to 77, wherein saidnucleic acid encoding an SHR polypeptide encodes any one of the proteinslisted in Table A or is a portion of such a nucleic acid, or a nucleicacid capable of hybridising with such a nucleic acid.

Item 79: Method according to any preceding Item 73 to 78, wherein saidnucleic acid sequence encodes an orthologue or paralogue of any of theproteins given in Table A.

Item 80: Method according to any one of Items 73 or 75 to 79, whereinsaid enhanced yield-related traits comprise increased yield, preferablyincreased biomass and/or increased seed yield relative to controlplants.

Item 81: Method according to any one of Items 77 to 80, wherein saidnucleic acid is operably linked to a constitutive promoter, preferablyto a GOS2 promoter, most preferably to a GOS2 promoter from rice.

Item 82: Method according to any preceding Item 73 to 81, wherein saidnucleic acid encoding an SHR polypeptide is of plant origin, preferablyfrom a dicotyledonous plant, further preferably from the familyBrassicaceae, more preferably from the genus Arabidopsis, mostpreferably from Arabidopsis thaliana.

Item 83: Plant or part thereof, including seeds, obtainable by a methodaccording to any preceding Item 73 to 82, wherein said plant or partthereof comprises a recombinant nucleic acid encoding an SHRpolypeptide.

Item 84: Construct comprising:

(i) nucleic acid encoding an SHR polypeptide as defined in Item 75;(ii) one or more control sequences capable of driving expression of thenucleic acid sequence of (a); and optionally(iii) a transcription termination sequence.

Item 85: Construct according to Item 84, wherein one of said controlsequences is a constitutive promoter, preferably a GOS2 promoter, mostpreferably a GOS2 promoter from rice.

Item 86: Use of a construct according to Item 84 or 85 in a method formaking plants having increased yield, particularly increased biomassand/or increased seed yield relative to control plants.

Item 87: Use of a construct according to Item 84 or 85 in a method formaking plants having increased TKW.

Item 88: Plant, plant part or plant cell transformed with a constructaccording to Item 84 or 85.

Item 89: Method for the production of a transgenic plant having enhancedyield-related traits relative to control plants, comprising:

(i) introducing and expressing in a plant a nucleic acid encoding an SHRas defined in Item 75; and(ii) cultivating the plant cell under conditions of reduced nutrientavailability.

Item 90: Method for the production of a transgenic plant havingincreased TKW relative to control plants, comprising:

(i) introducing and expressing in a plant a nucleic acid encoding an SHRas defined in Item 75; and(ii) cultivating the plant cell under non-nutrient limiting conditions.

Item 91: Products derived from a plant according to Item 83 or 88 and/orfrom harvestable parts of a plant according to Item 83 or 88.

Item 92: Use of a nucleic acid encoding an SHR polypeptide in enhancingyield-related traits, particularly in increasing seed yield and/or shootbiomass in plants, relative to control plants.

Item 93: Use of a nucleic acid encoding an SHR polypeptide in increasingTKW in plants, relative to control plants.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone. Thefollowing examples are not intended to completely define or otherwiselimit the scope of the invention.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

Example 1 Identification of Sequences Related to the Nucleic AcidSequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid used in the present invention was used forthe TBLASTN algorithm, with default settings and the filter to ignorelow complexity sequences set off. The output of the analysis was viewedby pairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

List A1 provides nucleic acid sequences related to SEQ ID NO: 1, andList A2 provides nucleic acid sequences related to SEQ ID NO: 3.

The expression “List A1” as used herein are equivalent andinterexchangeable with “Table A1”.

The expression “List A2” as used herein are equivalent andinterexchangeable with “Table A2”.

The term “table A” used in this specification is to be taken to specifythe content of table A1, table A2, table A3, table A4, and/or table A5.

The term “table A1” used in this specification is to be taken to specifythe content of table A1.

The term “table A2” used in this specification is to be taken to specifythe content of table A2.

The term “table A3” used in this specification is to be taken to specifythe content of table A3.

The term “table A4” used in this specification is to be taken to specifythe content of table A4.

The term “table A5” used in this specification is to be taken to specifythe content of table A5.

In one preferred embodiment, the term “table A” means table A1. Inanother preferred embodiment, the term “table A” means table A2. Inanother preferred embodiment, the term “table A” means table A3. Inanother preferred embodiment, the term “table A” means table A4. Inanother preferred embodiment, the term “table A” means table A5.

The term “table B” used in this specification is to be taken to specifythe content of table B1, table B2, table B3, and/or table B4.

The term “table B1” used in this specification is to be taken to specifythe content of table B1.

The term “table B2” used in this specification is to be taken to specifythe content of table B2.

The term “table B3” used in this specification is to be taken to specifythe content of table B3.

The term “table B4” used in this specification is to be taken to specifythe content of table B4.

In one preferred embodiment, the term “table B” means table B1. Inanother preferred embodiment, the term “table B” means table B2. Inanother preferred embodiment, the term “table B” means table B3. Inanother preferred embodiment, the term “table B” means table B4.

TABLE A1 Sequences related to SEQ ID NO: 1 Nucleic acid SEQ Protein SEQName Plant Source ID NO: ID NO: Ms_TCP_sugar Medicago sativa 1 2 AtTCP7Arabidopsis thaliana 7 8 OsTCP4 Oryza sativa 9 10 OsTCP10 Oryza sativa11 12 Pt\TCP Populus trichocarpa 13 14 Sl\TCP Solanum 15 16 lycopersicumVv\CAO70167 Vitis vinifera 17 18

TABLE A2 Sequences related to SEQ ID NO: 3 Nucleic acid SEQ Protein SEQName Plant Source ID NO: ID NO: Mt_TCP2_sugar Medicago truncatula 3 4Am\TCP\CAE45599 Antirrhinum majus 19 20 AT3G47620 Arabidopsis thaliana21 22 AtTCP15 Arabidopsis thaliana 23 24 Gh\TCP\AAD48836 Gossipumhirsutum 25 26 OSTCP12 Oryza sativa 27 28 OsTCP5 Oryza sativa 29 30Pt\TCP\scaff_124.66\ Populus trichocarpa 31 32 [1298]\f\[31-1218]Sd\TCP\AAT38718 Solanum demissum 33 34 Vv\TCP\AAD48836 Vitis vinifera 3536 Vv\TCP\CAO62540 Vitis vinifera 37 38

Concerning Epsin-like sequences, Table A3 provides a list of nucleicacid sequences related to the nucleic acid sequence used in the methodsof the present invention.

TABLE A3 Examples of Epsin-like polypeptides: Nucleic acid Protein PlantSource SEQ ID NO: SEQ ID NO: Arabidopsis thaliana 43 44 Arabidopsisthaliana 65 66 Vitis vinifera 67 68 Oryza sativa 69 70 Oryza sativa 7172 Avena fatua 73 74 Medicago truncatula 75 76 Arabidopsis thaliana 7778 Arabidopsis thaliana 79 80 Arabidopsis thaliana 81 82 Arabidopsisthaliana 83 84 Oryza sativa 85 86 Arabidopsis thaliana 87 88 Vitisvinifera 89 90 Arabidopsis thaliana 91 92 Arabidopsis thaliana 93 94Vitis vinifera 95 96 Chlamydomonas reinhardtii 97 98 Ostreococcuslucimarinus 99 100 Oryza sativa 101 102 Oryza sativa 103 Oryza sativa104 Oryza sativa 105 106 Oryza sativa 107 Oryza sativa 108 109 Oryzasativa 110 Brassica napus 111 112 Glycine max 113 114 Hordeum vulgare115 116 Medicago truncatula 117 118 Medicago truncatula 119 120Physcomitrella patents 121 122 Physcomitrella patents 123 124Physcomitrella patents 125 126 Populus trichocarpa 127 128 Populustrichocarpa 129 130 Populus trichocarpa 131 132 Solanum lycopersicum 133134 Triticum aestivum 135 136 Triticum aestivum 137 138 Arabidopsisthaliana 139 140 Zea mays 141 142

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

Concerning IPPT, table A4 provides a list of nucleic acid sequencesrelated to the nucleic acid sequence used in the methods of the presentinvention.

TABLE A4 Examples of IPPT polypeptide sequences, and encoding nucleicacid sequences: Nucleic acid Polypeptide Public database sequencesequence Name Source organism accession number SEQ ID NO: SEQ ID NO:Synec_IPPT Synechococcus sp. PCC 7942 U30252.3 143 144 Acama_IPPT (miaA)Acaryochloris marina MBIC11017 CP000828 145 146 Anava_IPPT Anabaenavariabilis ATCC 29413 CP000117 147 148 Glovi_IPPT Gloeobacter violaceusPCC 7421 BA000045 149 150 Micae_IPPT Microcystis aeruginosa PCC AM778958151 152 7806 Nossp_IPPT Nostoc sp. PCC 7120 DNA BA000019 153 154Proma1375_IPPT Prochlorococcus marinus subsp. AE017126 155 156 marinusstr. CCMP1375 Proma9211_IPPT Prochlorococcus marinus str. MIT CP000878157 158 9211 Proma9215_IPPT Prochlorococcus marinus str. MIT CP000825159 160 9215 Proma9301_IPPT Prochlorococcus marinus str. MIT CP000576161 162 9301 Proma9303_IPPT Prochlorococcus marinus str. MIT CP000554163 164 9303 Proma9312_IPPT Prochlorococcus marinus str. MIT CP000111165 166 9312 Proma9313_IPPT Prochlorococcus marinus BX572095 167 168MIT9313 Proma9515_IPPT Prochlorococcus marinus str. MIT CP000552 169 1709515 Proma9601_IPPT Prochlorococcus marinus str. CP000551 171 172 AS9601PromaMED4_IPPT Prochlorococcus marinus MED4 BX548174 173 174PromaNATL1A_IPPT Prochlorococcus marinus str. CP000553 175 176 NATL1APromaNATL2A_IPPT Prochlorococcus marinus str. CP000095 177 178 NATL2ASynecJA-3_IPPT Synechococcus sp. JA-3-3Ab CP000239 179 180 Synec307_IPPTSynechococcus sp. RCC307 CT978603 181 182 Synec6803_IPPT Synechocystissp. PCC 6803 BA000022 183 184 DNA Synec7803_IPPT Synechococcus WH7803CT971583 185 186 Synec8102_IPPT Synechococcus sp. WH8102 BX569689.1 187188 Synec9311_IPPT Synechococcus sp. CC9311 CP000435 189 190Synec9605_IPPT Synechococcus sp. CC9605 CP000110 191 192 Synec9902_IPPTSynechococcus sp. CC9902 CP000097 193 194 Theel_IPPT Thermosynechococcuselongatus BA000039 195 196 BP-1 Trier_IPPT Trichodesmium erythraeumCP000393 197 198 IMS101

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid sequence orpolypeptide sequence of interest. On other instances, special nucleicacid sequence databases have been created for particular organisms, suchas by the Joint Genome Institute.

Concerning SHR, table A5 provides a list of nucleic acid sequencesrelated to the nucleic acid sequence used in the methods of the presentinvention.

TABLE A5 Examples of SHR polypeptides Species of Nucleic acidPolypeptide Name origin SEQ ID NO: SEQ ID NO: Arabidopsis 208 209thaliana At4g37650 Arabidopsis 210 211 thaliana TA13018_3352 Pinus taeda212 213 22633_part Physcomitrella 214 215 patents 14911_partPhyscomitrella 216 217 patents Os03g31880 Oryza sativa 218 219Os07g39820 Oryza sativa 220 221 US200510879.113 Zea mays 222 223TA7750_4236 Lactuca sativa 224 225 AC147000 Medicago 226 227 truncatulaTC153082 Solanum 228 229 tuberosum WO2005001020_215 Eucalyptus 230 231grandis AM431974 Vitis vinifera 232 233 scaff_186.17 Populus 234 235trichocarpa TA2955_3988 Ricinus 236 237 communis US2004031072.68433Glycine max 238 239 CT027662 Medicago 240 241 truncatula

Example 2 Alignment of TCP Polypeptide Sequences

Alignment of polypeptide sequences was performed using the AlignXprogramme from the Vector NTI (Invitrogen) which is based on the popularClustal W algorithm of progressive alignment (Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res31:3497-3500). Default values are for the gap open penalty of 10, forthe gap extension penalty of 0,1 and the selected weight matrix isBlosum 62 (if polypeptides are aligned). Minor manual editing was doneto further optimise the alignment. The TCP1 polypeptides are aligned inFIG. 1 and the TCP2 polypeptides in FIG. 2.

A phylogenetic tree of TCP polypeptides (FIGS. 1 and 2) was constructedusing a neighbour-joining clustering algorithm as provided in the AlignXprogramme from the Vector NTI (Invitrogen).

Concerning Epsin-like sequences, default values are for the gap openpenalty of 10, for the gap extension penalty of 0,1 and the selectedweight matrix is Gonnet (if polypeptides are aligned). Minor manualediting may be done to further optimise the alignment. Sequenceconservation among Epsin-like polypeptides is essentially in theN-terminal ENTH domain of the polypeptides and in the C-terminal part,the central part usually being more variable in sequence length andcomposition. The Epsin-like polypeptides are aligned in FIG. 2.

Multiple sequence alignment of all the IPPT polypeptide sequences inTable A4 was performed using the AlignX algorithm (from Vector NTI 10.3,Invitrogen Corporation). Results of the alignment are shown in FIG. 3 ofthe present application. The N-terminal ATP/GTP-binding site motif A(P-loop) as represented by SEQ ID NO: 199, the Conserved motif IDSR(Q/L)(V/L/I) as represented by SEQ ID NO: 200, the Conserved motif II(N/D/S/T)(I/V)GTAKP(T/S) as represented by SEQ ID NO: 201, the Conservedmotif III L(V/A/I)GG(S/T)GLY as represented by SEQ ID NO: 202, and theConserved motif IV F/Y/L)AK(R/K/Q)Q(R/K/M)TWFR, are boxed. The putativezinc finger motif C2H2 (C-X2-C-X(12,18)-H-X5-H found in eukaryotictRNA-IPTs is marked with a bracket, and the conserved Cys and Hisresidues therein are boxed.

Concerning SHR, alignment of polypeptide sequences was performed usingthe AlignX programme from the Vector NTI (Invitrogen) which is based onthe popular Clustal W algorithm of progressive alignment (Thompson etal. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). NucleicAcids Res 31:3497-3500). Default values are for the gap open penalty of10, for the gap extension penalty of 0,1 and the selected weight matrixis Blosum 62 (if polypeptides are aligned). Minor manual editing wasdone to further optimise the alignment.

Regarding SHR-sequences, a phylogenetic tree of GRAS polypeptides (FIG.14) was constructed. A neighbour-joining tree of GRAS and SHR proteinswas constructed using GRAS proteins from rice, Arabidopsis andSHR-related proteins from the various organisms, were aligned usingMUSCLE. A neighbour-joining tree was produced with CLUSTALX. Bootstrapanalysis was performed for 100 iterations. The bootstrap support isshown only for the main nodes. The SHR related proteins are indicated.A. thaliana: Arabidopsis thaliana; E. grandis: Eucalyptus grandis; G.max: Glycine max; L. sativa: Latuca sativa; M. trucatula: Medicagotruncatula; O. sativa: Oryza sativa; P. taeda: Pinus taeda; P. patens:Physcomitrella patens; P. trichocarpa: Populus trichocarpa; R. communis:Ricinus communis; S. tuberosum: Solanum tuberosum; V. vinifera: Vitisvinifera; Z. mays: Zea mays;—part: partial sequence.

Example 3 Calculation of Global Percentage Identity Between PolypeptideSequences Useful in Performing the Methods of the Invention

Concerning TCP1 or TCP2, global percentages of similarity and identitybetween full length polypeptide sequences useful in performing themethods of the invention were determined using one of the methodsavailable in the art, the MatGAT (Matrix Global Alignment Tool) software(BMC Bioinformatics. 2003 4:29. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. CampanellaJ J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGATsoftware generates similarity/identity matrices for DNA or proteinsequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments using the Myers and Millerglobal alignment algorithm (with a gap opening penalty of 12, and a gapextension penalty of 2), calculates similarity and identity using forexample Blosum 62 (for polypeptides), and then places the results in adistance matrix. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line.

Parameters used in the comparison were:Scoring matrix: Blosum62

First Gap: 12 Extending gap: 2

Results of the software analysis are shown in Table B for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given below the diagonal in bold andpercentage similarity is given above the diagonal (normal face).

Table B: MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences.

TABLE B1 Ms_TCP_SUGAR family (TCP1) 1 2 3 4 5 6 7 1. Sl\TCP 46.4 45.140.7 47.2 29.9 33.3 2. pt\TCP 55.4 55.6 49.8 58.3 37.3 40.3 3.Vv\CAO70167 63.2 62.8 55.9 53.1 46.8 46.2 4. Ms_TCP_SUGAR 55.6 64.3 65.055.1 38.4 39.0 5. AtTCP7 57.6 66.9 61.6 70.0 37.2 39.1 6. OsTCP10 47.149.4 60.7 50.4 50.8 62.0 7. OsTCP4 50.2 50.6 59.2 53.0 53.2 73.5

TABLE B2 Mt_TCP2_SUGAR family (TCP2) 1 2 3 4 5 6 7 8 9 10 11 12  1.Am\TCPCAE45599 61.5 46.4 46.4 48.5 48.5 43.3 40.9 44.1 45.2 47.9 48.2 2. Vv\TCP\CAO62540 73.9 46.4 46.4 62.2 50.7 40.6 47.4 47.5 47.0 53.654.3  3. Sd\TCPAAT38718 56.8 59.1 100.0 39.9 34.9 35.9 35.7 35.9 34.463.2 64.5  4. Gh\TCP\AAD48836 56.8 59.1 100.0 39.9 34.9 35.9 35.7 35.934.4 63.2 64.5  5. Vv\TCP\CAO48409 59.3 74.4 58.0 58.0 48.9 38.3 49.847.5 44.7 43.1 44.5  6. Mt_TCP2_SUGAR 63.9 65.4 46.9 46.9 60.1 37.0 40.444.3 43.8 35.8 36.6  7. AtTCP14 54.6 53.4 45.6 45.6 46.2 52.4 33.5 38.039.9 35.5 36.6  8. AtTCP15 57.3 61.6 56.0 56.0 64.0 55.5 45.4 43.0 39.636.1 38.5  9. OsTCP12 59.0 60.5 49.6 49.6 58.4 57.5 48.9 51.9 67.0 34.637.0 10. OsTCP5 60.0 60.2 46.3 46.3 54.4 62.5 50.5 50.0 75.6 34.8 36.411. Pt197953_gw1.IV.3042.1 57.0 63.8 74.5 74.5 62.3 49.0 43.8 54.2 47.346.3 88.4 12. Pt266526_gw1.124.176.1 58.3 63.8 75.2 75.2 61.6 48.8 44.654.5 47.8 45.9 93.2

A MATGAT table for local alignment of a specific domain, or data on %identity/similarity between specific domains may also be constructed.

Concerning Epsin-like sequences, global percentages of similarity andidentity between full length polypeptide sequences useful in performingthe methods of the invention were determined using one of the methodsavailable in the art, the MatGAT (Matrix Global Alignment Tool) software(BMC Bioinformatics. 2003 4:29. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. CampanellaJ J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGATsoftware generates similarity/identity matrices for DNA or proteinsequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments using the Myers and Millerglobal alignment algorithm (with a gap opening penalty of 12, and a gapextension penalty of 2), calculates similarity and identity using forexample Blosum 62 (for polypeptides), and then places the results in adistance matrix. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line.

Concerning Epsin-like sequences, parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B3 for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given above the diagonal in bold andpercentage similarity is given below the diagonal (normal face).

The percentage identity between the Epsin-like polypeptide sequencesuseful in performing the methods of the invention can be as low as 14%amino acid identity compared to SEQ ID NO: 44.

TABLE B3 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13  1.SEQID2 96.4 59.5 46.1 41.0 40.9 39.5 37.9 43.0 25.0 25.4 25.4 30.7  2.CAB87689 96.5 58.2 45.1 39.9 40.1 38.4 37.2 42.0 25.7 25.5 25.5 30.9  3.CAO43767 73.4 71.6 46.7 42.0 41.5 40.9 39.4 43.8 25.4 27.8 27.8 31.1  4.CAD41810 61.3 61.3 61.8 44.3 67.3 42.4 40.2 95.8 26.6 25.7 25.7 28.8  5.BAD87030 58.8 59.2 58.6 58.9 40.8 88.0 82.2 42.8 24.8 25.0 25.0 27.5  6.AAB68030 56.1 56.1 55.3 76.0 57.5 40.5 39.3 64.3 26.8 26.9 26.9 29.3  7.EAZ13473 57.5 56.3 58.3 58.1 91.2 56.4 91.1 40.8 23.9 23.8 23.8 27.2  8.EAY75756 54.2 53.7 55.7 58.1 89.2 55.3 93.5 38.6 23.2 24.3 24.0 27.7  9.EAY95411 58.5 58.5 59.4 96.3 56.5 73.2 55.6 55.7 25.2 24.2 24.2 26.8 10.ABN08674 36.8 36.6 37.2 35.8 37.8 38.1 36.4 35.8 34.4 50.0 50.1 38.0 11.BAF01674 39.1 38.4 40.7 38.1 38.9 38.5 37.3 38.0 36.6 62.7 99.9 72.1 12.NP_850387 39.1 38.4 40.7 38.1 38.9 38.4 37.3 37.7 36.6 62.7 100.0 72.213. BAD44158 42.3 43.7 45.8 43.5 47.2 43.8 46.9 44.4 41.5 45.9 72.2 72.214. AAN72258 39.6 38.9 40.7 38.3 38.8 38.4 37.2 37.5 36.9 62.5 99.8 99.872.1 15. BAD19387 36.7 36.3 37.1 36.7 37.5 37.6 36.6 36.5 35.2 58.3 58.358.3 42.0 16. EAZ25008 36.7 36.3 37.1 36.7 37.5 37.6 36.6 36.5 35.2 58.358.3 58.3 42.0 17. CAB91599 33.3 33.6 34.6 34.6 34.5 35.2 34.5 34.1 33.559.2 70.5 70.5 50.9 18. CAO45312 37.4 38.2 39.1 38.3 39.7 40.5 38.6 38.936.8 58.3 63.6 63.6 47.8 19. AAL24360 33.5 33.6 34.9 34.8 34.5 35.2 34.534.0 33.7 59.1 70.4 70.4 50.9 20. AAC64305 34.1 33.4 35.3 31.6 30.7 28.231.7 30.7 29.5 24.9 29.5 29.5 41.3 21. CAN66991 33.8 34.0 36.7 35.9 35.635.4 33.8 34.8 35.0 56.3 58.7 58.7 49.7 22. XP001701452 38.9 39.7 38.136.9 37.1 35.4 38.9 38.7 35.2 29.4 29.1 29.1 38.9 23. XP001419857 20.520.3 20.5 18.7 18.5 18.2 19.3 18.8 17.5 12.4 13.5 13.5 18.9 14 15 16 1718 19 20 21 22 23  1. SEQID2 25.9 25.2 25.3 22.7 27.0 22.7 23.9 22.623.2 14.3  2. CAB87689 26.0 24.5 24.6 22.9 27.0 22.9 23.6 22.1 23.8 14.0 3. CAO43767 27.8 25.5 25.5 24.1 27.0 24.2 25.5 22.8 24.0 13.9  4.CAD41810 25.6 24.9 25.1 23.8 25.8 23.9 22.7 22.2 22.1 13.1  5. BAD8703025.0 25.1 25.1 22.5 25.7 22.6 21.2 21.5 23.1 12.9  6. AAB68030 26.9 25.525.5 24.1 26.3 24.2 20.7 23.3 22.7 12.2  7. EAZ13473 23.8 23.5 23.5 23.225.1 23.3 21.9 20.3 23.9 13.6  8. EAY75756 24.0 24.0 24.0 22.9 25.0 22.920.8 20.3 22.3 13.3  9. EAY95411 24.1 23.5 23.8 22.4 24.4 22.4 20.5 21.020.8 11.8 10. ABN08674 49.9 43.5 43.5 47.7 47.6 47.3 21.3 44.5 19.1 8.111. BAF01674 99.7 46.1 46.0 62.3 50.1 62.2 29.3 43.8 20.1 8.7 12.NP_850387 99.8 46.0 45.9 62.4 50.2 62.3 29.3 43.9 20.1 8.7 13. BAD4415872.1 33.6 33.6 44.6 38.7 44.6 41.0 35.9 24.8 12.2 14. AAN72258 45.7 45.662.2 50.2 62.1 29.2 43.8 20.1 8.7 15. BAD19387 58.1 99.9 40.8 44.7 40.720.1 40.7 20.7 8.0 16. EAZ25008 58.1 100.0 40.7 44.6 40.6 20.1 40.6 20.78.0 17. CAB91599 70.3 53.9 53.9 43.3 99.9 21.8 38.4 17.9 8.2 18.CAO45312 63.7 56.8 56.8 54.9 43.2 22.6 77.7 20.6 8.9 19. AAL24360 70.253.8 53.8 99.9 54.5 21.8 38.3 17.9 8.3 20. AAC64305 29.4 23.9 23.9 23.925.7 23.9 18.9 16.2 26.5 21. CAN66991 58.7 52.9 52.9 50.6 81.8 50.6 23.719.5 8.7 22. XP001701452 29.1 30.7 30.7 26.6 31.3 26.6 26.2 31.7 14.223. XP001419857 13.5 12.4 12.4 11.6 13.5 11.6 43.1 12.9 20.3

Concerning IPPT, global percentages of similarity and identity betweenfull length polypeptide sequences useful in performing the methods ofthe invention were determined using one of the methods available in theart, the MatGAT (Matrix Global Alignment Tool) software (BMCBioinformatics. 2003 4:29. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. CampanellaJ J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGATsoftware generates similarity/identity matrices for DNA or proteinsequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments using the Myers and Millerglobal alignment algorithm (with a gap opening penalty of 12, and a gapextension penalty of 2), calculates similarity and identity using forexample Blosum 62 (for polypeptides), and then places the results in adistance matrix. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B4 for the globalsimilarity and identity over the full length of the polypeptidesequences (excluding the partial polypeptide sequences).

The percentage identity between the full length polypeptide sequencesuseful in performing the methods of the invention can be as low as 39%amino acid identity compared to SEQ ID NO: 144.

TABLE B4 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences of Table A4. 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16  1. Synec_IPPT 55 54 42 51 53 41 42 41 41 45 42 44 39 4139  2. Acama_IPPT 70 65 48 58 66 39 42 40 42 43 42 42 41 41 42  3.Anava_IPPT 71 76 49 66 94 39 41 41 42 43 43 43 41 42 43  4. Globa_IPPT65 64 63 45 49 35 40 36 37 39 37 38 37 38 37  5. Micae_IPPT 67 72 76 6264 43 39 42 42 42 44 42 42 43 42  6. Nossp_IPPT 70 76 96 63 76 39 42 4041 43 42 43 40 41 42  7. Proma1375_IPPT 64 64 62 60 63 61 60 53 54 56 5457 53 53 54  8. Proma9211_IPPT 61 60 63 61 61 62 79 52 53 58 53 57 51 5052  9. Proma9215_IPPT 61 60 61 59 65 60 75 73 87 50 83 50 70 88 71 10.Proma9301_IPPT 62 61 60 59 63 60 76 73 94 50 85 49 70 88 71 11.Proma9303_IPPT 62 60 63 58 61 63 74 75 70 70 49 98 49 50 50 12.Proma9312_IPPT 64 63 64 60 66 64 74 75 91 93 71 49 71 84 72 13.Proma9313_IPPT 62 59 62 57 61 61 74 74 69 70 99 71 49 49 49 14.Proma9515_IPPT 61 61 61 58 62 59 73 70 84 83 67 82 68 70 84 15.Proma9601_IPPT 62 59 63 59 63 62 75 71 94 95 70 91 70 82 70 16.PromaMED4_IPPT 61 62 61 60 63 61 76 71 86 84 69 84 69 92 84 17.PromaNATL1A_IPPT 61 62 63 60 61 62 75 72 68 70 70 72 70 72 69 70 18.PromaNATL2A_IPPT 61 61 61 59 61 60 74 71 67 70 69 72 69 71 69 70 19.Synec307_IPPT 61 62 62 60 61 60 70 69 67 68 74 69 72 66 68 66 20.Synec6803_IPPT 70 72 73 63 76 73 65 63 66 63 63 64 63 61 64 62 21.Synec7803_IPPT 61 57 59 55 62 59 70 70 64 64 76 64 76 64 63 64 22.Synec8102_IPPT 67 62 63 60 61 65 73 71 64 65 83 66 81 64 66 66 23.Synec9311_IPPT 62 60 60 61 63 60 72 71 66 66 79 67 78 67 66 67 24.Synec9605_IPPT 65 61 63 60 61 63 73 76 66 67 82 69 80 66 67 67 25.Synec9902_IPPT 66 61 63 59 61 63 74 75 66 66 80 69 79 67 65 68 26.Synecsp_IPPT 62 60 60 60 60 59 54 53 52 52 54 54 53 49 53 50 27.Theel_IPPT 66 72 70 60 67 70 63 58 59 61 62 61 61 58 59 58 28.Trier_IPPT 69 72 79 63 73 79 63 62 61 60 59 63 58 60 61 61 29.Escco_miaA 57 55 53 57 57 53 54 55 49 50 54 50 53 51 52 51 30.Arath_IPT2 37 35 35 35 36 35 33 32 33 34 33 34 32 33 34 33 31.Sacce_MOD5_IPPT 38 36 36 39 40 35 36 35 33 34 33 35 33 34 34 36 32.Homsa_IPPT 36 36 35 35 39 36 36 36 34 34 35 36 34 33 34 34 17 18 19 2021 22 23 24 25 26 27 28 29 30 31 32  1. Synec_IPPT 40 41 44 52 42 44 4246 45 46 49 53 39 20 22 20  2. Acama_IPPT 38 38 44 60 40 42 43 42 44 4959 58 35 21 22 23  3. Anava_IPPT 40 39 46 62 40 43 42 43 45 49 56 65 3520 21 23  4. Globa_IPPT 40 39 44 46 38 41 41 41 41 46 48 45 37 21 22 19 5. Micae_IPPT 39 39 43 61 42 42 43 41 43 46 52 59 35 21 23 23  6.Nossp_IPPT 41 40 44 62 41 44 42 43 45 47 55 64 35 21 21 23  7.Proma1375_IPPT 54 54 50 44 52 54 52 55 56 36 40 40 33 19 22 21  8.Proma9211_IPPT 54 53 48 42 53 52 52 56 55 35 38 40 35 17 21 19  9.Proma9215_IPPT 51 50 46 43 46 47 47 47 47 34 40 41 31 18 19 19 10.Proma9301_IPPT 52 52 46 43 47 47 47 48 47 36 41 40 32 18 21 20 11.Proma9303_IPPT 53 51 58 46 64 65 67 64 65 41 45 41 37 19 21 20 12.Proma9312_IPPT 54 53 47 44 48 48 48 49 49 36 41 42 32 17 20 20 13.Proma9313_IPPT 53 51 58 46 64 65 66 64 65 40 45 41 37 19 21 20 14.Proma9515_IPPT 56 55 44 42 47 46 46 49 48 36 41 39 31 18 21 19 15.Proma9601_IPPT 52 51 45 43 46 47 47 47 47 36 40 41 31 18 20 21 16.PromaMED4_IPPT 53 53 46 45 47 49 48 50 48 35 40 40 31 19 22 19 17.PromaNATL1A_IPPT 94 50 42 47 51 49 52 51 35 40 39 35 19 22 20 18.PromaNATL2A_IPPT 96 50 43 49 51 50 52 51 36 40 39 34 19 22 20 19.Synec307_IPPT 68 67 45 55 57 58 58 56 40 46 41 36 20 21 21 20.Synec6803_IPPT 63 61 61 43 47 43 46 45 46 51 61 35 21 22 20 21.Synec7803_IPPT 65 64 68 61 68 71 66 66 39 41 39 35 19 21 19 22.Synec8102_IPPT 71 70 75 64 78 67 79 76 41 43 43 36 18 23 20 23.Synec9311_IPPT 68 68 70 65 81 80 66 68 41 43 41 37 20 21 19 24.Synec9605_IPPT 71 69 72 63 78 87 80 78 41 45 44 37 18 20 19 25.Synec9902_IPPT 72 71 71 63 77 87 80 87 39 44 44 37 19 21 20 26.Synecsp_IPPT 52 51 54 58 53 54 56 55 53 50 45 35 19 25 25 27. Theel_IPPT59 59 59 65 60 60 62 61 60 62 51 36 20 22 23 28. Trier_IPPT 62 60 57 7360 62 62 61 63 59 69 35 20 21 21 29. Escco_miaA 54 52 51 54 52 54 56 5453 51 54 54 22 21 22 30. Arath_IPT2 33 32 32 35 33 33 34 32 33 34 35 3538 25 28 31. Sacce_MOD5_IPPT 37 36 33 37 34 35 35 33 37 38 36 37 37 4732 32. Homsa_IPPT 35 34 35 35 33 35 34 35 36 38 38 38 39 55 51

Concerning SHR, global percentages of similarity and identity betweenfull length polypeptide sequences useful in performing the methods ofthe invention is determined using one of the methods available in theart, the MatGAT (Matrix Global Alignment Tool) software (BMCBioinformatics. 2003 4:29. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. CampanellaJ J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGATsoftware generates similarity/identity matrices for DNA or proteinsequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments using the Myers and Millerglobal alignment algorithm (with a gap opening penalty of 12, and a gapextension penalty of 2), calculates similarity and identity using forexample Blosum 62 (for polypeptides), and then places the results in adistance matrix.

Parameters used in the comparison are:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Example 4 Identification of Domains Comprised in Polypeptide SequencesUseful in Performing the Methods of the Invention

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Panther, ProDom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 44 are presented in Table C1.

TABLE C1 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 44. Amino acidcoordinates Accession Accession on SEQ Database number name ID NO 44InterPro IPR001026 Epsin, N-terminal HMMPfam PF01417 ENTH 25-148HMMSmart SM00273 ENTH 26-152 ProfileScan PS50942 ENTH 20-152 InterProIPR008943 Phosphoinositide-binding clathrin adaptor, N-terminalsuperfamily SSF48473 PI_bind_N 25-238

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 144 are presented in Table C2.

TABLE C2 InterPro scan results of the polypeptide sequence asrepresented by SEQ ID NO: 144 Integrated Integrated database InterProaccession database accession Integrated database accession number andname name number name IPR002627 BlastProDom PD004674 MIAA_SYNP7_Q8GIT6;tRNA isopentenyltransferase IPR002627 HMMPfam PF01715.6 IPP transferasetRNA isopentenyltransferase IPR002627 HMMTigr TIGR00174 miaA: tRNAdelta(2)- tRNA isopentenylpyrophosphate isopentenyltransferase IPR011593BlastProDom PD005388 MIAA_SYNP7_Q8GIT6 Isopentenyl transferase-like IPRnon-integrated tmhmm PTHR11088 TRNA DELTA(2)- ISOPENTENYLPYROPHOSPHATETRANSFERASE-RELATED IPR non-integrated superfamily SSF52540 P-loopcontaining nucleoside triphosphate hydrolases

Example 5 Topology Prediction of the Polypeptide Sequences Useful inPerforming the Methods of the Invention

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters are selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark

Concerning SEQ ID NO:44, a number of parameters were selected, such asorganism group (non-plant or plant), cutoff sets (none, predefined setof cutoffs, or user-specified set of cutoffs), and the calculation ofprediction of cleavage sites (yes or no).

The results of TargetP 1.1 analysis of the polypeptide sequence asrepresented by SEQ ID NO: 44 are presented Table D. The “plant” organismgroup has been selected, no cutoffs defined, and the predicted length ofthe transit peptide requested. The subcellular localization of thepolypeptide sequence as represented by SEQ ID NO: 44 may be thecytoplasm or nucleus, no transit peptide is predicted.

TABLE D TargetP 1.1 analysis of the polypeptide sequence as representedby SEQ ID NO: 44 Length (AA) 560 Chloroplastic transit peptide 0.105Mitochondrial transit peptide 0.100 Secretory pathway signal peptide0.168 Other subcellular targeting 0.872 Predicted Location / Reliabilityclass 2 Predicted transit peptide length /

Example 6 Assay Related to the Polypeptide Sequences Useful in

Performing the Methods of the Invention

The polypeptide sequence as represented by SEQ ID NO: 2 or SEQ ID NO: 4is a transcription factor with DNA binding activity. The ability of atranscription factor to bind to a specific DNA sequence can be tested byelectrophoretic mobility shift assays (EMSAs; also called gelretardation assays), which is well known in the art, and reportedspecifically for TCPs by Kosugi & Ohashi (2002) Plant J 30: 337-348, andby Li et al. (2005) PNAS 102(36): 12978-83. Also reported by Kosugi &Ohashi are methods to detect dimerization partners and specificity,using for example, the yeast two-hybrid system, while Li et al. describechromatin immunoprecipitation experiments to characterize the promotersto which TCPs bind to.

Concerning Epsin-like polypeptides, lipid binding may be performed asdescribed by Hom et al. (2007). Solutions of phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine (Avanti) andPhosphatidylinositol(4,5) bisphosphate diC16 (C16-PtdIns(4,5)P2, EchelonBiosciences Inc.) dissolved in CHCl3/MeOH/H2O (65:25:4, by volume) weremixed and dried down under vacuum. The lipids were resuspended in 50 mMTris, 100 mM KCl (pH 7.0) and incubated at 64° C. for 1 h. The liposomeswere then frozen in liquid nitrogen and thawed at 37° C. for threecycles. The liposome solution was passed through an Avanti extruder toproduce 1.0 μm liposomes. Liposomes were collected by centrifugation at25,000 g for 10 min and resuspended to a final concentration of 2 mMtotal lipids in 100 μl 20 mM Tris, 100 mM KCl buffer (pH 6.0, 7.0 or8.0). Liposomes were incubated with the GST-fusion ENTH and ANTH domainsor GST (2-5 μg/ml final protein concentration) for 30 min at roomtemperature and then collected again by centrifugation. The liposomepellets were resuspended in 100 μl of buffer and analyzed by SDS-PAGEand Coomassie brilliant blue staining for the presence of lipid-bindingproteins.

Concerning IPPT, polypeptides useful in performing the methods of theinvention display IPPT activity. Many assays exist to measure such IPPTactivity, including complementation assays of a yeast strain withdefective endogenous IPPT activity (encoded by the MOD 5 gene; Golovkoet al. (2002) Plant Molec Biol 49: 161-169), complementation assays ofan E. coli strain with defective endogenous IPPT activity (encoded bythe miaA gene; Dihanich et al. (1987) Mol Cell Biol 7: 177-184), orquantification of cytokinins in tRNA (Gray et al. (1996) Plant Physiol110: 431-438, Miyawaki et al. (2006) Proc Natl Acad SCi USA 103(44):16598-16603). A person skilled in the art is well aware of suchexperimental procedures to measure IPPT activity, including IPPTactivity of an IPPT polypeptide as represented by SEQ ID NO: 144.

Example 7 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention

Cloning of the TCP Nucleic Acid Sequences

The nucleic acid sequences used in the methods of the invention wasamplified by PCR using as template a custom-made Medicago cDNA library(in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed usingHifi Taq DNA polymerase in standard conditions, using 200 ng of templatein a 50 μl PCR mix. The primers used were:

TCP1-sense (SEQ ID NO: 39):5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGTCTAACCACAAGGAAACA 3′TCP1-reverse, complementary (SEQ ID NO: 40):5′GGGGACCACTTTGTACAAGAAAGCTGGGTGAATAAAGTACAAAACACCGAA 3′TCP2-sense (SEQ ID NO: 41): 5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGGAATTGGAAGGTGATCAT 3′TCP2-reverse, complementary (SEQ ID NO: 42): 5′GGGGACCACTTTGTACAAGAAAGCTGGGTTCAGATCATACACTTCTAATTGCTT 3′which include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, pTCP1or pTCP2. Plasmid pDONR201 was purchased from Invitrogen, as part of theGateway° technology.

The entry clone comprising SEQ ID NO: 1 or SEQ ID NO: 2 was then used inan LR reaction with a destination vector used for Oryza sativatransformation. This vector contained as functional elements within theT-DNA borders: a plant selectable marker; a screenable marker expressioncassette; and a Gateway cassette intended for LR in vivo recombinationwith the nucleic acid sequence of interest already cloned in the entryclone. A rice GOS2 promoter or an HMGP promoter for constitutiveexpression was located upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vector wastransformed into Agrobacterium strain LBA4044 according to methods wellknown in the art.

Cloning of Epsin-Like Sequences

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used were

prm09481 (SEQ ID NO: 46; sense, start codon in bold):5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggatttcatgaaggtcttc-3′ andprm09482 (SEQ ID NO: 47; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggttcacagacaatttcactgctt-3′,which include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”,pEpsin-like. Plasmid pDONR201 was purchased from Invitrogen, as part ofthe Gateway® technology.

The entry clone comprising SEQ ID NO: 43 was then used in an LR reactionwith a destination vector used for Oryza sativa transformation. Thisvector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 45) for root specific expression was locatedupstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::Epsin-like (FIG. 4) was transformed into Agrobacterium strainLBA4044 according to methods well known in the art.

Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO: 143

Unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in (Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994),Current Protocols in Molecular Biology, Current Protocols. Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfax (1993) by R. D. D. Croy, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

The Synechococcus sp. PCC 7942 nucleic acid sequence encoding an IPPTpolypeptide sequence as represented by SEQ ID NO: 144 was amplified byPCR using as template genomic DNA extracted Synechococcus sp. PCC 7942.The following primers, which include the AttB sites for Gatewayrecombination, were used for PCR amplification:

1) Prm 07646 (SEQ ID NO: 206, sense):5′- ggggacaagtttgtacaaaaaagcaggcttaaacaatggaatcgcgtttgaaacc-3′2) Prm 07645 (SEQ ID NO: 207, reverse, complementary):5′- ggggaccactttgtacaagaaagctgggttcaaacgccctcactctttcg-3′

PCR was performed using Hifi Taq DNA polymerase in standard conditions.A PCR fragment of the expected length (including attB sites) wasamplified and purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombined in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Cloning of the SHR Nucleic Acid Sequence (SEQ ID NO: 208)

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used were

(SEQ ID NO: 243; sense, start codon in bold):5′-ggggacaagtttgtacaaaaaagcaggcttaaacaa tggatactctctttagactagtca-3′ and(SEQ ID NO: 244; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggtaaataaaaacaacccttt acg-3′,which include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, pSHR.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway°technology.

The entry clone comprising SEQ ID NO: 208 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 242) for constitutive expression was locatedupstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::SHR (FIG. 16) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

Example 8 Expression Vector Construction Using the Nucleic Acid Sequenceas Represented by SEQ ID NO: 143

The entry clone comprising SEQ ID NO: 143 was subsequently used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A ricedehydrin promoter (SEQ ID NO: 204) for seed-specific expression waslocated upstream of this Gateway cassette. A second destination vectorfor Oryza sativa transformation was also produced, with a riceproteinase inhibitor promoter (SEQ ID NO: 205) also for seed-specificexpression.

After the LR recombination step, the resulting expression vectorspDehydrin::IPPT and pProt_inhib::IPPT (FIG. 4) for seed-specificexpression, were independently transformed into Agrobacterium strainLBA4044 according to methods well known in the art.

Example 9 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M patent U.S. Pat. No. 5,164,310. Severalcommercial soybean varieties are amenable to transformation by thismethod. The cultivar Jack (available from the Illinois Seed foundation)is commonly used for transformation. Soybean seeds are sterilised for invitro sowing. The hypocotyl, the radicle and one cotyledon are excisedfrom seven-day old young seedlings. The epicotyl and the remainingcotyledon are further grown to develop axillary nodes. These axillarynodes are excised and incubated with Agrobacterium tumefacienscontaining the expression vector. After the cocultivation treatment, theexplants are washed and transferred to selection media. Regeneratedshoots are excised and placed on a shoot elongation medium. Shoots nolonger than 1 cm are placed on rooting medium until roots develop. Therooted shoots are transplanted to soil in the greenhouse. T1 seeds areproduced from plants that exhibit tolerance to the selection agent andthat contain a single copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/I) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/1 BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown DCW and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Cotton Transformation (Concerning TCP1/TCP2 and Epsin-Like Sequences)

Cotton is transformed using Agrobacterium tumefaciens according to themethod described in U.S. Pat. No. 5,159,135. Cotton seeds are surfacesterilised in 3% sodium hypochlorite solution during 20 minutes andwashed in distilled water with 500 μg/ml cefotaxime. The seeds are thentransferred to SH-medium with 50 μg/ml benomyl for germination.Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cmpieces and are placed on 0.8% agar. An Agrobacterium suspension (approx.108 cells per ml, diluted from an overnight culture transformed with thegene of interest and suitable selection markers) is used for inoculationof the hypocotyl explants. After 3 days at room temperature andlighting, the tissues are transferred to a solid medium (1.6 g/lGelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg etal., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/mlcefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria.Individual cell lines are isolated after two to three months (withsubcultures every four to six weeks) and are further cultivated onselective medium for tissue amplification (30° C., 16 hr photoperiod).Transformed tissues are subsequently further cultivated on non-selectivemedium during 2 to 3 months to give rise to somatic embryos. Healthylooking embryos of at least 4 mm length are transferred to tubes with SHmedium in fine vermiculite, supplemented with 0.1 mg/l indole aceticacid, 6 furfurylaminopurine and gibberellic acid. The embryos arecultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the2 to 3 leaf stage are transferred to pots with vermiculite andnutrients. The plants are hardened and subsequently moved to thegreenhouse for further cultivation.

Cotton Transformation (Concerning IPPT)

Cotton (Gossypium hirsutum L.) transformation is performed usingAgrobacterium tumefaciens, on hypocotyls explants. The commercialcultivars such as Coker 130 or Coker 312 (SeedCo, Lubbock, Tex.) arestandard varieties used for transformation, but other varieties can alsobe used. The seeds are surface sterilized and germinated in the dark.Hypocotyl explants are cut from the germinated seedlings to lengths ofabout 1-1.5 centimeter. The hypotocyl explant is submersed in theAgrobacterium tumefaciens inoculum containing the expression vector, for5 minutes then co-cultivated for about 48 hours on MS+1.8 mg/l KNO3+2%glucose at 24° C., in the dark. The explants are transferred the samemedium containing appropriate bacterial and plant selectable markers(renewed several times), until embryogenic calli is seen. The calli areseparated and subcultured until somatic embryos appear. Plantletsderived from the somatic embryos are matured on rooting medium untilroots develop. The rooted shoots are transplanted to potting soil in thegreenhouse. T1 seeds are produced from plants that exhibit tolerance tothe selection agent and that contain a single copy of the T-DNA insert.

Example 10 Phenotypic Evaluation Procedure

9.1 Evaluation Setup

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

In case of a confirmation round, four T1 events were further evaluatedin the T2 generation following the same evaluation procedure as for theT1 generation but with more individuals per event. From the stage ofsowing until the stage of maturity the plants were passed several timesthrough a digital imaging cabinet. At each time point digital images(2048×1536 pixels, 16 million colours) were taken of each plant from atleast 6 different angles.

Four T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

Drought Screen (Concerning TCP1/TCP2 and SHR)

Concerning TCP1/TCP2, plants from T2 seeds were grown in potting soilunder normal conditions until they approached the heading stage.

Concerning SHR, plants from T2 seeds are grown in potting soil undernormal conditions until they approached the heading stage.

They were then transferred to a “dry” section where irrigation waswithheld. Humidity probes were inserted in randomly chosen pots tomonitor the soil water content (SWC). When SWC went below certainthresholds, the plants were automatically re-watered continuously untila normal level was reached again. The plants were then re-transferredagain to normal conditions. The rest of the cultivation (plantmaturation, seed harvest) was the same as for plants not grown underabiotic stress conditions. Growth and yield parameters are recorded asdetailed for growth under normal conditions.

Drought Screen (Epsin-Like Sequences)

Plants from T2 seeds are grown in potting soil under normal conditionsuntil they approach the heading stage. They are then transferred to a“dry” section where irrigation is withheld. Humidity probes are insertedin randomly chosen pots to monitor the soil water content (SWC). WhenSWC goes below certain thresholds, the plants are automaticallyre-watered continuously until a normal level is reached again. Theplants are then re-transferred again to normal conditions. The rest ofthe cultivation (plant maturation, seed harvest) is the same as forplants not grown under abiotic stress conditions. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Drought Screen (IPPT)

Plants from a selected number of events are grown in potting soil undernormal conditions until they approached the heading stage. They are thentransferred to a “dry” section where irrigation is withheld. Humidityprobes are inserted in randomly chosen pots to monitor the soil watercontent (SWC). When SWC go below certain thresholds, the plants areautomatically re-watered continuously until a normal level is reachedagain. The plants are then re-transferred to normal conditions. The restof the cultivation (plant maturation, seed harvest) is the same as forplants not grown under abiotic stress conditions. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Nitrogen Use Efficiency Screen (Concerning TCP1/TCP2)

Rice plants from T2 seeds are grown in potting soil under normalconditions except for the nutrient solution. The pots were watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) wasthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Nitrogen Use Efficiency Screen (Concerning Epsin-Like Sequences)

Rice plants from T2 seeds are grown in potting soil under normalconditions except for the nutrient solution. The pots are watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) isthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Nitrogen Use Efficiency Screen (Concerning SHR)

Rice plants from T2 seeds were grown in potting soil under normalconditions except for the nutrient solution. The pots were watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) wasthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Salt Stress Screen (Concerning Epsin-Like Sequences)

Plants are grown on a substrate made of coco fibers and argex (3 to 1ratio). A normal nutrient solution is used during the first two weeksafter transplanting the plantlets in the greenhouse. After the first twoweeks, 25 mM of salt (NaCl) is added to the nutrient solution, until theplants are harvested. Seed-related parameters are then measured.

Salt Stress Screen (Concerning IPPT)

Plants are grown on a substrate made of coco fibers and argex (3 to 1ratio). A normal nutrient solution is used during the first two weeksafter transplanting the plantlets in the greenhouse. After the first twoweeks, 25 mM of salt (NaCl) is added to the nutrient solution, until theplants were harvested. Growth and yield parameters are recorded asdetailed for growth under normal conditions.

Reduced Nutrient (Nitrogen) Availability Screen (Concerning IPPT)

Plants from six events (T2 seeds) are grown in potting soil under normalconditions except for the nutrient solution. The pots are watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) isthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

9.2 Statistical Analysis: F Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF test. A significant F test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

Because two experiments with overlapping events were carried out, acombined analysis was performed. This is useful to check consistency ofthe effects over the two experiments, and if this is the case, toaccumulate evidence from both experiments in order to increaseconfidence in the conclusion. The method used was a mixed-model approachthat takes into account the multilevel structure of the data (i.e.experiment—event—segregants). P values were obtained by comparinglikelihood ratio test to chi square distributions.

9.3 Parameters Measured

Biomass-Related Parameter Measurement

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the area measured at the time point at which theplant had reached its maximal leafy biomass. The early vigour is theplant (seedling) aboveground area three weeks post-germination. Increasein root biomass is expressed as an increase in total root biomass(measured as maximum biomass of roots observed during the lifespan of aplant); or as an increase in the root/shoot index (measured as the ratiobetween root mass and shoot mass in the period of active growth of rootand shoot).

Early vigour was determined by counting the total number of pixels fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point fromdifferent angles and was converted to a physical surface value expressedin square mm by calibration. The results described below are for plantsthree weeks post-germination.

Seed-Related Parameter Measurements

The mature primary panicles were harvested, counted, bagged,barcode-labelled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand KernelWeight (TKW) is extrapolated from the number of filled seeds counted andtheir total weight. The Harvest Index (HI) in the present invention isdefined as the ratio between the total seed yield and the above groundarea (mm²), multiplied by a factor 10⁶. The total number of flowers perpanicle as defined in the present invention is the ratio between thetotal number of seeds and the number of mature primary panicles. Theseed fill rate as defined in the present invention is the proportion(expressed as a %) of the number of filled seeds over the total numberof seeds (or florets).

Example 11 Results of the Phenotypic Evaluation of the Transgenic Plants

The results of the evaluation of transgenic rice plants expressing aTCP1 or TCP2 nucleic are shown below. The % difference is transgenicplants compared to corresponding nullizygotes.

Results of the Evaluation of Rice Plants Expressing ConstructpHMGP::TCP1(Medicago sativa) or pGOS2::TCP1 (Medicago sativa) UnderNon-Stress and Drought Conditions

Drought Non-stress pHMGP::TCP1 pHMGP::TCP1 Total seed 42% 11% weight No.filled seeds 43% 9% Fill rate 22% Flowers per 7% 4% panicle No. firstpanicles 7% Harvest index 36% 7% Aboveground <4% area Emergence 8%vigour TKW <5%

A positive tendency was noticed in the following parameters: emergencevigour, total seed weight and TKW for construct pGOS2::TCP1 (Medicagosativa) under non-stress conditions.

Results of the Evaluation of Rice Plants Expressing Construct orpGOS2::TCP2 (Medicago truncatula) Under Non-Stress and DroughtConditions

Drought Non-stress Parameter pGOS2::TCP1 pGOS2::TCP1 Harvest Index 21%9% No. Filled Seeds 23% 5% Fill rate Na 6% Root-Shoot Na 9% index Totalweight 27% <5%  seeds No. Flowers per 10% Na panicle TKW <5% Na No.first panicles  8% Na

The results of the evaluation of transgenic rice plants expressing anEpsin-like nucleic acid are presented below. An increase of more than 5%was observed for total seed number, total seed yield, number of filledseeds, and fill rate. In addition, an increase of more than 5% inaboveground biomass and in early vigour was observed in both T1 and T2generations for at least one event

TABLE E Yield increase observed in plants expressing the Epsin-likenucleic acid of SEQ ID NO: 44: T2 T1 P-value Overall % Overall %combined Parameter increase P-value increase analysis Total weight ofseeds >5 0.0011 >5 0.0023 Total number of seeds >5 0.033 >5 0.1068Number of filled seeds >5 0.0017 >5 0.0069 Fill rate >5 0.0024 2.70.0001

Results of the Phenotypic Evaluation of the Transgenic Rice PlantsExpressing the Nucleic Acid Sequence Encoding an IPPT Polypeptide asRepresented by SEQ ID NO: 144, Under the Control of a DehydrinSeed-Specific Promoter

The results of the evaluation of T1 and T2 generation transgenic riceplants expressing the nucleic acid sequence encoding an IPPT polypeptideas represented by SEQ ID NO: 144, under the control of a dehydrinseed-specific promoter, and grown under normal growth conditions, arepresented below.

There was a significant increase in the early vigor, in the abovegroundbiomass, in the total seed yield per plant, in the total number ofseeds, in the number of filled seeds, in the number of flowers perpanicle, and in the harvest index of the transgenic plants compared tocorresponding nullizygotes (controls), as shown in Table F

TABLE F Results of the evaluation of T1 and T2 generation transgenicrice plants expressing the nucleic acid sequence encoding an IPPTpolypeptide as represented by SEQ ID NO: 144, under the control of adehydrin promoter for seed-specific expression. Overall average %Overall average % increase in 6 events increase in 4 events Trait in theT1 generation in the T2 generation Early vigor 25 25 Aboveground biomass2 8 Total seed yield per plant 14 13 Total number of seeds 8 15 Totalnumber of filled seeds 15 13 Harvest index 14 5 Number of first panicles13 3

Results of the Phenotypic Evaluation of the Transgenic Rice PlantsExpressing the Nucleic Acid Sequence Encoding an IPPT Polypeptide asRepresented by SEQ ID NO: 144, Under the Control of a ProteinaseInhibitor Seed-Specific Promoter

The results of the evaluation of T1 generation transgenic rice plantsexpressing the nucleic acid sequence encoding an IPPT polypeptide asrepresented by SEQ ID NO: 144, under the control of a proteinaseinhibitor seed-specific promoter, and grown under normal growthconditions, are presented below.

There was a significant increase in the early vigor, in the abovegroundbiomass, in the total seed yield per plant, in the total number ofseeds, in the number of filled seeds, and in the number of flowers perpanicle, of the transgenic plants compared to corresponding nullizygotes(controls), as shown in Table G.

TABLE G Results of the evaluation of T1 generation transgenic riceplants expressing the nucleic acid sequence encoding an IPPT polypeptideas represented by SEQ ID NO: 144, under the control of a proteinaseinhibitor promoter for seed-specific expression. Overall average %increase in the two Trait best events in the T1 generation Early vigor34 Aboveground biomass 15 Total seed yield per plant 20 Total number ofseeds 23 The results of the evaluation of transgenic rice plantsexpressing an SHR nucleic acid under non-stress conditions are presentedbelow. Parameter % difference over controls TKW 7.3%

The results of the evaluation of transgenic rice plants expressing anSHR nucleic acid under conditions of reduced nitrogen availability arepresented below.

Parameter % difference over controls Aboveground area 10.2% Emergencevigour 23.2% Root biomass 23.6% Fill rate 25.3% TKW   7%

1. A method for enhancing a yield-related trait in a plant relative to acontrol plant, comprising: a) modulating expression of a nucleic acidencoding a TCP2 polypeptide in a plant, and optionally selecting for aplant having an increased yield-related trait relative to a controlplant; b) modulating expression of a nucleic acid encoding an Epsin-likepolypeptide in a plant, and optionally selecting for a plant having anincreased yield-related trait relative to a control plant, wherein saidEpsin-like polypeptide comprises an ENTH domain; c) modulatingexpression of a nucleic acid encoding a tRNAdelta(2)-isopentenylpyrophosphate transferase (IPPT) polypeptide in aseed of a plant, and optionally selecting for a plant having anincreased yield-related trait relative to a control plant, wherein saidIPPT polypeptide comprises (i) a tRNA isopentenyltransferase domain withan InterPro accession IPR002627, and (ii) an N-terminal ATP/GTP-bindingsite motif A (P-loop); or d) modulating expression of a nucleic acidencoding an SHR polypeptide in a plant, and optionally selecting for aplant having an increased yield-related trait relative to a controlplant, wherein said plant is grown under conditions of sub-optimalnutrient availability.
 2. The method of claim 1, wherein said increasedyield-related trait is obtained by modulating expression of a nucleicacid encoding a TCP2 polypeptide in a plant, wherein said TCP2polypeptide comprises: (i) a TCP domain having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the TCPdomain of any of the sequences indicated in FIG. 2; (ii) a domain havingat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moresequence identity to Domain 1 of any of the sequences indicated in FIG.2; (iii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more sequence identity to Domain 2 of any of thesequences indicated in FIG. 2; and (iv) a domain having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity toDomain 3 of any of the sequences indicated in FIG.
 2. 3. The method ofclaim 2, wherein said TCP2 polypeptide further comprises: (v) a domainhaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moresequence identity to Domain 4 of any of the sequences indicated in FIG.2; and (vi) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more sequence identity to Domain 5 of any of thesequences indicated in FIG.
 2. 4. The method of claim 1, wherein thenucleic acid encodes an orthologue or paralogue of SEQ ID NO:
 4. 5. Themethod of claim 2, wherein said modulated expression is increasedexpression of a nucleic acid encoding a TCP2 polypeptide.
 6. The methodof claim 5, wherein said increased expression is effected by any one ormore of T-DNA activation tagging, TILLING, or homologous recombination.7. The method of claim 5, wherein said increased expression is effectedby introducing and expressing in a plant a nucleic acid sequenceencoding said TCP2 polypeptide.
 8. The method of claim 1, wherein saidenhanced yield-related traits comprise increased seed weight relative toa control plant.
 9. The method of claim 7, wherein said nucleic acid isoperably linked to a constitutive promoter, a HMGP (High Mobility GroupProtein) promoter, or a GOS2 promoter.
 10. The method of claim 7,wherein said nucleic acid encoding a TCP2 polypeptide is of plantorigin, or wherein said nucleic acid is from a plant of the Medicagofamily.
 11. A plant or part thereof including seeds obtained by themethod of claim 1, or a progeny of said plant, wherein said plant orpart thereof, or said progeny, comprises a nucleic acid transgeneencoding the TCP2 polypeptide.
 12. A construct comprising: (i) a nucleicacid sequence encoding a TCP2 polypeptide as defined in claim 2; (ii)one or more control sequences capable of driving expression of thenucleic acid of (i); and optionally (iii) a transcription terminationsequence.
 13. The construct of claim 12, wherein said one or morecontrol sequences is at least a constitutive promoter, an HMGP, or aGOS2 promoter.
 14. A method for obtaining a plant having increased yieldor seed yield relative to a control plant comprising growing a plantwhich comprises the construct of claim
 12. 15. A plant, plant part, orplant cell transformed with the construct of claim 12, or a progeny ofsaid plant, wherein said progeny comprises said construct.
 16. A methodfor the production of a transgenic plant having increased seed yieldrelative to a control plant, which method comprises: (i) introducing andexpressing in a plant or plant cell a nucleic acid encoding a TCP2polypeptide as defined in claim 2; (ii) cultivating the plant or plantcell under conditions promoting plant growth and development; and (iii)optionally selecting a plant having increased seed yield relative to acontrol plant.
 17. A transgenic plant having increased yield orincreased seed yield relative to a control plant resulting fromincreased expression of a nucleic acid encoding a TCP2 polypeptide asdefined in claim 2, or a transgenic plant cell or progeny derived fromsaid transgenic plant, wherein said transgenic plant cell or progenycomprises the nucleic acid encoding the TCP2 polypeptide.
 18. Thetransgenic plant of claim 17, wherein said increased seed yield is oneor more of the following: (i) increased seed weight; (ii) increasedharvest index; (iii) increased Thousand Kernel Weight, (iv) increasednumber of flowers per panicle, (v) increased fill rate, or (vi)increased number of filled seeds.
 19. The transgenic plant of claim 17,wherein said plant is a crop plant, a monocot or a cereal, or atransgenic plant cell derived from said plant.
 20. Harvestable parts ofthe transgenic plant of claim 17, wherein said harvestable partscomprise seeds having the nucleic acid transgene encoding the TCP2polypeptide.
 21. Products derived from the transgenic plant of claim 17and/or from harvestable parts of said plant, wherein the productscomprise said nucleic acid transgene encoding a TCP2 polypeptide.